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Cyclotides are plant cyclic peptides with exceptional stability and diverse bioactivity, making them promising candidates for biomedical applications. Therefore, the study of cyclotides has attracted increasing attention in recent years. However, the existing cyclotide detection methods face limitations in sensitivity, accuracy, and reliability. To address these challenges, we developed an integrated strategy using a combination of strong cation exchange chromatography techniques for removing interfering small molecules, Orbitrap Exploris 480 mass spectrometry (OEMS); this is a detection and database searching-based method for cyclotide verification, which greatly improved the sensitivity, accuracy, and reliability of cyclotide identification. This strategy was subsequently employed for cyclotide mapping in Viola with a minute amount of starting tissue, resulting the identification of 65 known and 18 potentially novel cyclotides, which is the largest dataset of cyclotides for Viola philippica . This strategy provided valuable insights into the cyclotide diversity and distribution in V. philippica , with potential applications in drug discovery and other biomedical fields.

Bacteria threaten human and animal health, and standard antibiotics no longer effective. Antibiotic-resistant microorganisms can make infection treatment challenging and perhaps fail. Investigating the attributes of cyclotide, a peptide with promising antibacterial properties that holds great potential in the field of antibiotic research. The structure of these cyclic peptides involves six conserved cysteine residues that form three disulfide bonds, resulting in a cyclic cystine knot (CCK). This feature guarantees their durability when exposed to changes in temperature, chemicals, and enzymatic degradation. The two cyclotides, cycloviolacin O17 and mra30, were obtained from Viola dalatensis Gadnep through a series of techniques including the use of a 50% acetonitrile/49% miliQ water/1% formic acid solution for extraction, ammonium salt precipitation, RP-HPLC purification and sequence identification by LC-MS/MS. These cyclotides exhibit antibacterial effects on specific strains of bacteria like Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa at a concentration of 0.2 mg/mL, leading to inhibition zones ranging from 10 to 14 mm. In addition, the disulfide bonds play a crucial role in the antibacterial function of cyclotides. Disrupting the disulfide bonds through ankylation reaction results in the loss of antibacterial properties in the cyclotides (cyO17 and mra30). The minimum inhibitory concentration (MIC) values of mra30 and cyO17 are significantly low, ranging from 0.1 to 0.6 µM. These values are approximately three times lower than the MIC values observed in salt precipitation samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cyclotides are a type of defense peptide most commonly found in the Violaceae family of plants, exhibiting various biological activities. In this study, we focused on the Viola japonica as our research subject and conducted transcriptome sequencing and analysis using high-throughput transcriptomics techniques. During this process, we identified 61 cyclotides, among which 25 were previously documented, while the remaining 36 were designated as vija 1 to vija 36. Mass spectrometry detection showed that 21 putative cyclotides were found in the extract of V. japonica. Through isolation, purification and tandem mass spectrometry, we characterized and investigated the activities of five cyclotides. Our results demonstrated inhibitory effects of these cyclotides on the growth of Acinetobacter baumannii and Bacillus subtilis, with minimum inhibitory concentrations (MICs) of 4.2 μM and 2.1 μM, respectively. Furthermore, time killing kinetic assays revealed that cyclotides at concentration of 4 MICs achieved completely bactericidal effects within 2 h. Additionally, fluorescence staining experiments confirmed that cyclotides disrupt microbial membranes. Moreover, cytotoxicity studies showed that cyclotides possess cytotoxic effects, with IC 50 values ranging from 0.1 to 3.5 μM. In summary, the discovery of new cyclotide sequences enhances our understanding of peptide diversity and the exploration of their activity lays the foundation for a deeper investigation into the mechanisms of action of cyclotides., (© 2024. The Author(s).)

Pansy and viola edible flowers were grown hydroponically with different levels of Mg and Mn. The nutritional composition was determined using standard methods. Free sugars, fatty acids, organic acids, tocopherols, and phenolic compounds were analyzed using various HPLC and GC devises. The extract's antimicrobial, antioxidant, cytotoxicity, and anti-inflammatory activity were assessed. The results indicated that Mg enrichment negatively affected plant growth and mineral accumulation but improved photosynthetic performance. The edible flowers contained significant amounts of protein, low levels of fat, and varying sugar contents, such as glucose and fructose. Various fatty acids and phenolic compounds were identified, with different concentrations depending on the treatment. The flowers exhibited antioxidant potential, antimicrobial activity, cytotoxic effects, and anti-inflammatory properties. The correlations between the investigated parameters not only expand knowledge on Mg and Mn interaction but also catalyze significant advancements in sustainable agriculture and food health, fostering a healthier and more conscious future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cyclotides, plant-derived cysteine-rich peptides, exhibit a wide range of beneficial biological activities and possess exceptional structural stability. Cyclotides are commonly distributed throughout the Violaceae family. Viola dalatensis Gagnep, a Vietnamese species, has not been well studied, especially for cyclotides. This pioneering research explores cyclotides from V. dalatensis as antimicrobials. This study used a novel approach to enhance cyclotides after extraction. The approach combined 30% ammonium sulfate salt precipitation and RP-HPLC. A comprehensive analysis was performed to ascertain the overall protein content, flavonoids content, polyphenol content, and free radical scavenging capacity of compounds derived from V. dalatensis. Six known cyclotides were sequenced utilizing MS tandem. Semi-purified cyclotide mixtures (M1, M2, and M3) exhibited antibacterial efficacy against Bacillus subtilis (inhibitory diameters: 19.67-23.50 mm), Pseudomonas aeruginosa (22.17-23.50 mm), and Aspergillus flavus (14.67-21.33 mm). The enriched cyclotide precipitate from the stem extract demonstrated a minimum inhibitory concentration (MIC) of 0.08 mg/mL against P. aeruginosa, showcasing significant antibacterial effectiveness compared to the stem extract (MIC: 12.50 mg/mL). Considerable advancements have been achieved in the realm of cyclotides, specifically in their application as antimicrobial agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Viola tricolor is a medicinal plant with documented application as an anti-inflammatory herb. The standard of care for the treatment of inflammatory bowel disease is immunosuppressive therapeutics or biologics, which often have undesired effects. We explored V. tricolor herbal preparations that are rich in an emerging class of phytochemicals with drug-like properties, so-called cyclotides. As an alternative to existing inflammatory bowel disease medications, cyclotides have immunomodulatory properties, and their intrinsic stability allows for application in the gastrointestinal tract, for instance, via oral administration. We optimized the isolation procedure to improve the yield of cyclotides and compared the cellular effects of violet-derived organic solvent-extracts, aqueous preparations, and an isolated cyclotide from this plant on primary human T lymphocytes and macrophages, i.e., cells that are crucial for the initiation and progression of inflammatory bowel disease. The hot water herbal decoctions have a stronger immunosuppressive activity towards proliferation, interferon- γ , and interleukin-21 secretion of primary human T cells than a DCM/MeOH cyclotide-enriched extract, and the isolated cyclotide kalata S appears as one of the active components responsible for the observed effects. This effect was increased by a longer boiling duration. In contrast, the DCM/MeOH cyclotide-enriched extract was more effective in reducing the levels of cytokines interleukin-6, interleukin-12, interleukin-23, tumor necrosis factor- α , and C - X-C motif chemokine ligand 10, secreted by human monocyte-derived macrophages. Defined cyclotide preparations of V. tricolor have promising pharmacological effects in modulating immune cell responses at the cytokine levels. This is important towards understanding the role of cyclotide-containing herbal drug preparations for future applications in immune disorders, such as inflammatory bowel disease., Competing Interests: The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commecial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Over the past two decades, microfluidic-based separations have been used for the purification, isolation, and separation of biomolecules to overcome difficulties encountered by conventional chromatography-based methods including high cost, long processing times, sample volumes, and low separation efficiency. Cyclotides, or cyclic peptides used by some plant families as defense agents, have attracted the interest of scientists because of their biological activities varying from antimicrobial to anticancer properties. The separation process has a critical impact in terms of obtaining pure cyclotides for drug development strategies. Here, for the first time, a mimic of the high-performance liquid chromatography (HPLC) on microfluidic chip strategy was used to separate the cyclotides. In this regard, silica gel-C18 was synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance ( 1 H-NMR) and then filled inside the microchannel to prepare an HPLC C18 column-like structure inside the microchannel. Cyclotide extract was obtained from Viola ignobilis by a low voltage electric field extraction method and characterized by HPLC and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). The extract that contained vigno 1, 2, 3, 4, 5, and varv A cyclotides was added to the microchannel where distilled water was used as a mobile phase with 1 µL/min flow rate and then samples were collected in 2-min intervals until 10 min. Results show that cyclotides can be successfully separated from each other and collected from the microchannel at different periods of time. These findings demonstrate that the use of microfluidic channels has a high impact on the separation of cyclotides as a rapid, cost-effective, and simple method and the device can find widespread applications in drug discovery research., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

There are 500 species of Viola(Violaceae) worldwide, among which 111 species are widely distributed in China and have a long medicinal history and wide varieties. According to the authors' statistics, a total of 410 compounds have been isolated and identified from plants of this genus, including flavonoids, terpenoids, phenylpropanoids, organic acids, nitrogenous compounds, sterols, saccharides and their derivatives, volatile oils and cyclotides. The medicinal materials from these plants boast anti-microbial, anti-viral, anti-oxidant and anti-tumor activities. This study systematically reviewed the chemical constituents and pharmacological activities of Viola plants to provide a basis for further research and clinical application.

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2022. Published by Elsevier B.V.)

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-1β, 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., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

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.

Ethnopharmacological Relevance: Viola yedoensis Makiho (VY, Violaceae) is a well-known medicinal herb in Chinese medicine, which is traditionally used to treat inflammation-related disorders, including allergic skin reactions. Although studies have uncovered its anti-inflammatory effects and corresponding bioactive constituents, the exact mechanism of action is still unclear in treating allergic skin reactions., Objective: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by severe pruritus, dry, edema and inflamed skin. It affects people's quality of life seriously and causes huge economic losses to society. This study proposes VY as a possible remedy for atopic dermatitis since its traditional usage and superior anti-inflammatory effects., Materials and Methods: Atopic dermatitis-like skin lesion was induced by topical application of 2,4-dinitrochlorobenzene (DNCB) in ICR mice. After treatment with Viola yedoensis Makiho ethanol extract (VYE) or dexamethasone (positive control) for 3 weeks, skin pathological observation and the molecular biological index were performed for therapeutic evaluation, including visual inspection in the change of the stimulated skin, scar formation, pathological morphology by hematoxylin and eosin (HE) staining, the measurement of interleukin IL-1β, IL-6 and tumor necrosis factor-alpha (TNF-α) levels in serum as well as spleen index. The expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were analyzed by western blot. The ratio of CD4+/CD8+ T lymphocyte in the spleen was detected by flow cytometry. Meanwhile, immunohistochemistry staining for CD68 identified the number of activated macrophages in skin lesions. Additionally, a reliable ultrahigh-performance liquid chromatography coupled with a Q exactive hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) method was established for the systematic identification and characterization of main components in VYE., Results: VYE alleviated DNCB-stimulated AD-like lesions symptoms as evidenced by a significant decrease in hypertrophy, hyperkeratosis, and infiltration of inflammatory cells in dorsal skin. The levels of IL-1β, IL-6, and TNF-α in serum were suppressed in mice treated with VYE as compared to the DNCB-induced model group. Also, the administration of VYE reduced the ratio of CD4+/CD8+ T lymphocyte in the spleen and the number of activated macrophages stimulated by DNCB. Besides, the expression of iNOS and COX-2 were down-regulated in the dorsal skin., Conclusions: VYE showed therapeutic effects on atopic dermatitis in DNCB-induced AD-like lesion mouse models by inhibiting the T cell-mediated allergic immune response. Our results indicated that VY could act as a potential remedy for atopic dermatitis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In the present study, the phytase enzyme was purified from Lactobacillus plantarum with a 3.08% recovery, 9.57-purification fold, and with a specific activity of 278.82 EU/mg protein. Then, the effects of the 5 EU and 10 EU purified phytase was determined on the plant growth, quality, the macro-micro nutrient content of pansy (Viola × wittrockiana), which is of great importance in ornamental plants industry. The research was established under greenhouse conditions with natural light in 2017. The pansy seeds were coated with phytase enzyme solution, sown in a peat environment, and transferred to pots at the seedling period. In general, the 5 EU and 10 EU applications increase plant height, the number of leaves per plant, the number of side branches per plant, and flower height parameters compared to control. Also, micro- and macronutrient values in soil and plant samples were examined. According to the results, the phytase application on pansy cultivation positively affected the properties and yielded high quality of plants., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

The aim of the study was to investigate volatile compounds from the aerial parts of Viola canina L. of the family Violaceae grown wild in Northern Kazakhstan for the first time. Viola canina is a common and widespread species of the temperate northern hemisphere. The oil from the aerial parts of V. canina , was obtained by hydrodistillation and investigated by gas chromatography combined with mass spectrometry. The main component of V. canina essential oil was phytol (55.2%). The obtained results showed differences in the composition of already studied Viola essential oils, such as essential oil of V. odorata L., V. etrusca Erben, V. tricolor L., V. arvensis Murr., V. tianshanica Maxim., V. serpens Wall. and V. hamiltoniana D. Don. There is a certain similarity with the composition of the essential oil of V. arvensis where phytol was also found as one of the main component (11.4%).

Viola yedoensis Makino was used to treat inflammation, viral hepatitis, acute pyogenic infection, and ulcerative carbuncles. However, the protective effect on immunological liver injury (ILI) of V. yedoensis had been rarely reported. This study aimed to explore the protective effect of n-butanol extract (BE) from V. yedoensis on ILI in vitro and in vivo. In vitro, the BE significantly inhibited the secretions of Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg) in the HepG2.2.15 cells and the replication of HBV DNA. The research data in vivo revealed that the BE reduced the levels of alanine transaminase (ALT), aspartate transaminase (AST), and methane dicarboxylic aldehyde (MDA) in liver tissues of the ConA-induced mice, while increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and the effective contents of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and the BE could ameliorate liver histological lesions. These results motivated a further investigation into the chemical constituents of BE. Four coumarins (esculetin, prionanthoside, cichoriin, and esculin) and one flavonoid (quercetin-3-O-galactoside) were isolated from the BE by silica gel column chromatography and recrystallization, of which structures were eventually confirmed by 1 H-NMR, 13 C-NMR, and MS., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Viola odorata Linn or sweet violet has several biological activities due to the presence of flavonoids, tannins, alaloid, glycoside, and saponins. However, susceptibility of these compounds to harsh conditions and low solubility is a great challenge for their incorporation into food products. Therefore, encapsulation can be an effective approach in this respect. In the present study, chitosan-coated microcapsules loaded with Viola extract were prepared for the first time and the effects of independent variables (sodium alginate: 1-1.5%, calcium chloride: 0.6-1.5% and extract concentrations: 5-10%) on encapsulation efficiency (EE%) were investigated. After evaluation of the model, the optimum condition for preparation of microcapsules was selected as 1.47% sodium alginate, 5.02% extract and 1.42% CaCl 2 with EE% of 83.21%. The microcapsules developed at this condition had an acceptable spherical shape and the results obtained in Fourier transform-infrared spectroscopy (FTIR) indicated the presence of the extract within the microcapsules. The mean diameters of the uncoated and chitosan-coated microcapsules were 73 and 141 μm, respectively. The in vitro release in acidic medium (pH 1.5) and phosphate buffer saline (pH 7) were 43.21% and 95.39%, respectively. The prepared extract-loaded microcapsules have potential to be used in food products providing acceptable antioxidant activity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The metabolic syndrome increases the risk of different diseases such as type 2 diabetes. The prevalence of metabolic syndrome has rapidly grown and affected more than 230 million people worldwide. Viola odorata is a traditionally used plant for the treatment of diabetes; however, its mechanism to manage diabetes is still unknown., Purpose: This study was designed to systematically assess the mechanism of action of Viola odorata in diabetes., Methods: An extensive literature search was made to establish an ingredient-target database of Viola odorata . Of these, targets related to diabetes were identified and used to develop a protein-protein interaction network (PPIN) by utilizing the STITCH database. The obtained PPIN was assessed through Gene Ontology (GO) enrichment analysis based on ClueGO plugin., Results: According to the acquired data, there were about 143 chemical constituents present in Viola odorata having 119 protein targets. Of these, 31 targets were established to give the pharmacological effect against diabetes. The UniProt database was used for screening of 31 targets, out of which Homo sapiens contained 22 targets. Ultimately, 207 GO terms, grouped into 41 clusters, were found by gene analysis, and most of them were found to be linked with diabetes. According to findings, several proteins including TP53, BCL2, CDKN1A, 1L6, CCND1, CDKN2A, and RB1 have a significant role in the treatment of diabetes by Viola odorata ., Conclusion: The possible activity of Viola odorata in the management of diabetes may be mediated by several molecular mechanisms, including the glutamine metabolic process, IRE1-mediated unfolded protein response, and pentose metabolic process., Competing Interests: The authors declare that there is no conflict of interests., (Copyright © 2020 Manal Ali Buabeid et al.)

Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low-abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS 2 ) "fingerprint" ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS 2 spectral fingerprint ions cross referenced with CyBase-a cyclotide database-to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptides, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria . Coupling the observation of highly abundant MS 2 ions with mass shift analysis, we identify a new set of small, highly disulfide-bound cysteine-rich L. siceraria peptides.

Background: Mammospheres are breast cancer stem cells (BCSCs) that could be yielded through culturing cells in non-adherent and non-differentiating condition. With regard to therapy resistance of cancer stem cells (CSCs), it is essential to discover efficient approaches targeting CSCs. Viola odorata extract has been considered as a traditional herbal anti-metastatic drug in several cancer cells. Effect of this drug on BCSCs has not been clearly identified. Current study tries to detect and to compare effect of Viola odorata extract on malignant characterization of breast cancer cell lines and BCSCs., Materials and Methods: MCF7 and SKBR3 and their derived mammospheres as BCSCs were used and the effect of alcoholic extraction of Viola odorata on apoptosis and malignant characters of MCF7, SKBR3 and their derived BCSCs were analyzed and compared., Results: Viola odorata extract induced cell death in MCF7, SKBR3 and their derived mammospheres through apoptosis without any effects on MCF10A. Also, this extract showed anti-migratory, anti-invasion and anti-colony formation activity in MCF7, SKBR3 and their derived mammospheres which was significantly more in MCF7- and SKBR3-derived mammospheres. Also, this extract decreased size and volume of tumors generated by MCF7, SKBR3 and their derived mammospheres in chicken embryo model., Conclusion: Viola odorata extract exerted anti-cancerous activity on both breast cancer cell lines and their derived BCSCs. Anti-cancerous activity of this extract was significantly more in MCF7-, SKBR3-derived mammospheres in comparison with dedicated cell lines. Data suggest that Viola odorata extract mostly targets cancerous cells, not normal cells with exception in high concentration. It acts in a cell-dependent manner.

Viola tricolor Linn. is used as cardio-protective and anti-hypertensive agent in traditional medicine. Current study objective was to evaluate cardio-protective and hypotensive effects of Viola tricolor L. in vitro and in vivo studies. Viola tricolor L. crude extract (Vt.Cr) and its fractions (Aqueous and organic) were tested at rabbit atria and aorta coupled to Power Lab Data Acquisition System for cardio depressant and vasorelaxant effects in vitro whereas in vivo Blood Pressure was checked by invasive method in normotensive ketamine-diazepam anesthetized rats. Isoproterenol was employed for acute myocardial infarction (AMI) and left ventricular hypertrophy (LVH) development and cardioprotective effects of Vt.Cr were evaluated hemodynamically and histopathologically. Vt.Cr and its fractions decreased heart rate and contractile force in paired atria and relaxed Phenylephrine (1 µM) and K + (80 mM) stimulated contractions in aorta possibly mediated through Voltage dependent L-type calcium channels blockage supported by in vivo hypotensive action. In LVH, Vt.Cr lowered Angiotensin Converting Enzymes and renin, increased cyclic Guanosine Monophosphate and nitric oxide levels, decreased cardiomyocytes size and fibrosis attributed to Gallic acid as detected by High Performance Liquid Chromatography. Partial positive results were seen hemodynamically and histologically in AMI Viola tricolor L. showed vasorelaxant, cardio-relaxant, hypotensive, and cardio protective effect validating traditional practice in cardiovascular disorders., (© 2020 Federation of American Societies for Experimental Biology.)

Viola is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of cyclotides. Many Viola species are used as medicinal herbs across Asia and are often consumed by humans in teas for the treatment of diseases, including ulcers and asthma. Previous studies reported the isolation of cyclotides from Viola species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, Viola species from Vietnam have not been investigated to date. Here, the discovery of cyclotides from three Viola species ( V. arcuata , V. tonkinensis , and V. austrosinensis ) collected in the northern mountainous region of Vietnam is reported. Ten cyclotides were isolated from these three Viola species: four are novel and six were previously reported to be expressed in other plants. The structures of three of the new bracelet cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), the cancer cytotoxicity of the cyclotides isolated from V. arcuata was assessed. All tested cyclotides were cytotoxic against cancer cells, albeit to varying degrees. The sequences discovered in this study significantly expand the understanding of cyclotide diversity, especially in comparison with other cyclotides found in plants from the Asian region.

The present study aimed to evaluate the nutritional and bioactive potential of four edible flowers (borage, centaurea, camellia, and pansies). Significant differences were observed among the four. Water was the main constituent (>76%, fresh weight - fw). Linoleic and palmitic acids were the major fatty acids found in borage and red and yellow pansies, while in camellia it was the arachidic acid. In white pansies, behenic and arachidic acids were predominant. Concerning vitamin E, α-tocopherol was the major vitamer. Carotenoids values varied between 5.8 and 181.4 mg β-carotene/100 g dry weight (dw) in centaurea and borage, respectively, being particularly rich in lutein. Malic acid was the major organic acid, except in centaurea, where succinic acid was predominant. Fructose, glucose and sucrose were detected in all flowers. These results can contribute to the knowledge of these edible flowers and consequently increase their popularity among consumers and in the food industry., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Edible flowers are a new gourmet product; however, they are not always available all years. Thus, it is essential to find out technologies to guarantee this product for a longer time. Flowers of four species (borage [Borago officinalis], heartsease [Viola tricolor], kalanchoe [Kalanchoe blossfeldiana], and dandelion [Taraxacum officinale]) were subjected to freezing (in their natural form and in ice cubes) and analyzed in terms of visual appearance, the content of flavonoids, hydrolysable tannins, phenolics, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power), and microbial quality after storage for 1 and 3 months. Flowers in ice cubes showed similar appearance to fresh ones during the 3 months of storage, whereas frozen flowers were only equivalent up to 1 month with the exception of kalanchoe. Even though flowers in ice cubes showed good appearance after 3 months of storage, they had the lowest values of bioactive compounds and antioxidant activity. On the contrary, when frozen, the content of bioactive compounds maintained or even increased up to 1 month of storage compared to fresh flowers, except for borage. Furthermore, in both freezing treatments, the microorganisms' counts decreased or maintained when compared to fresh samples, except in dandelion. In general, both treatments may allow keeping the flowers after their flowering times. PRACTICAL APPLICATION: The market of edible flowers is increasing, although they are a very perishable product with short shelf-life. Edible flowers are stored in the cold (frozen or in ice cubes); however, the effect on the bioactive compounds and microbial quality that this treatment may have on borage (Borago officinalis), heartsease (Viola tricolor), kalanchoe (Kalanchoe blossfeldiana), and dandelion (Taraxacum officinale) flowers is unknown. So, the present study was conducted to increase the knowledge about the changes that freezing treatments may have in different edible flowers. The results of the present study underline that each flower has different behavior at frozen and ice cubes storage. However, freezing flowers maintain/increase the contents of bioactive compounds, while ice cubes not. Both treatments are effective in protecting flowers from microorganism growth. So, suggesting that both freezing treatments can be used as a preservative method and may allow keeping the flowers after their flowering times., (© 2020 Institute of Food Technologists®.)

Background: The antiprotozoal and antioxidant activities of Viola tricolor and Laurus nobilis have been reported recently. Thus, the existing study pursued to assess the growth inhibition effect of methanolic extract of V. tricolor (MEVT) and acetonic extract of L. nobilis (AELN) against five Babesia parasites and Theileria equi in vitro and in vivo., Results: MEVT and AELN suppressed Babesia bovis, B. bigemina, B. divergens, B. caballi, and T. equi growth at half-maximal inhibitory concentration (IC 50 ) values of 75.7 ± 2.6, 43.3 ± 1.8, 67.6 ± 2.8, 48 ± 3.8, 54 ± 2.1 μg/mL, and 86.6 ± 8.2, 33.3 ± 5.1, 62.2 ± 3.3, 34.5 ± 7.5 and 82.2 ± 9.3 μg/mL, respectively. Qualitative phytochemical estimation revealed that both extracts containing multiple bioactive constituents and significant amounts of flavonoids and phenols. The toxicity assay revealed that MEVT and AELN affected the mouse embryonic fibroblast (NIH/3 T3) and Madin-Darby bovine kidney (MDBK) cell viability with half-maximum effective concentrations (EC 50 ) of 930 ± 29.9, 1260 ± 18.9 μg/mL, and 573.7 ± 12.4, 831 ± 19.9 μg/mL, respectively, while human foreskin fibroblasts (HFF) cell viability was not influenced even at 1500 μg/mL. The in vivo experiment revealed that the oral administration of MEVT and AELN prohibited B. microti multiplication in mice by 35.1 and 56.1%, respectively., Conclusions: These analyses indicate the prospects of MEVT and AELN as good candidates for isolating new anti-protozoal compounds which could assist in the development of new drug molecules with new drug targets.

The cyclotides are the largest known family of cyclic proteins, which are found in several plant families including Violaceae. They are circular bioactive peptides consisting of 28-37 amino acids, which possess a cyclic cystine knot (CCK) motif and could be useful in biotechnology and drug design as scaffolds for peptide-based drugs. This study describes our finding of a potentially novel gene transcript from the petals of the Iranian Viola tricolor (V. tricolor) flowers. This study is based on the cDNA screening method employed for isolation of cyclotide precursor genes and in silico analysis. Our study resulted in the finding of a novel cyclotide-like precursor from V. tricolor, which is documented in the NCBI by GenBank accession number: KP065812. The in silico analysis revealed that there are lots of similar sequences in many other plant families and they all exhibit some different features from previously discovered cyclotide precursors. The differences occur particularly in the main cyclotide domain that exists without the usual CCK structure. All of these hypothetical precursors have a conserved ER-signal sequence, a Cysteine (C)-rich sequence forming two zinc finger motifs and a cyclotide-like region containing several conserved elements including two highly conserved C residues. In conclusion, using the cDNA screening method we found a potentially new cyclotide-like precursor gene and in silico studies revealed its significant characteristics that may open up a new research line on the distribution and evolution of cyclotides., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Finding non-systemic antipyretic option in cancer patients who simultaneously receive several other drugs seems be logical. This study was designed to evaluate complementary therapy with Viola odorata L. oil for fever control in febrile neutropenic children., Methods and Materials: In a randomized placebo controlled clinical trial, 41 febrile children were divided into two groups. Children in the active drug group received viola oil (20 drops) to be rubbed on the peripheral margin of the patient umbilicus. Primary outcome measure of the study was the mean axillary temperature in the 30, 60, and 240 minutes after the intervention., Results: The mean temperature reduced significantly in the viola group after 30 minutes of administration (p =0.005), while there was no significant change in the placebo group (p =1.00). The number of patients who received paracetamol as the rescue treatment was significantly lower in the viola group than that in the placebo group (5 vs. 17, p =0.001)., Conclusion: The results of our study showed the safety and efficacy of complementary therapy with Viola odorata L. oil for fever control in febrile neutropenic children during hospital course., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cation generates dehydroalanine at a single cysteine residue, which is easily identified by the odd-electron loss of ·SCH 2 CONH 2 . The presence of dehydroalanine in cyclotides provides a site-selective ring-opening pathway that, in turn, generates linear cyclotide analogues in the gas phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua , where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well-suited for cyclotide species that are not abundant enough to characterize with traditional methods.

Background: Dry eye disease is a disorder of the tear film. In this study, the effect of Viola odorata L. oily extract was examined for the treatment of patients suffering from dry eye disease., Methods: A randomised, double-blind, placebo-controlled study was designed. During the trial, Schirmer's test, tear breakup time, Oxford staining and the Ocular Surface Disease Index were assessed. Overall, 105 patients with dry eye symptoms between the ages of 18 and 60 years were allocated to the violet-almond oil, almond oil and placebo (1% w/v hydroxypropyl methylcellulose solution) groups. The treatment and placebo were administered intranasally, two drops three times a day for one month. The patients were followed up for four weeks. A total of 91 patients (32, 29 and 30 in the violet-almond oil, almond oil and placebo groups, respectively) completed the study., Results: At baseline, there was no difference between the three groups in terms of demographic data and the measurement parameters. After the intervention, the results revealed that the Schirmer's score without local anaesthesia and the tear breakup time results significantly improved in the violet-almond oil group. One-way ANOVA indicated a significant improvement in the Schirmer's score, tear breakup time and Ocular Surface Disease Index of the treatment group, as compared with the other groups (p < 0.05). However, the obtained results did not present any significant mean difference between and within the groups of the Oxford staining grade (p > 0.05)., Conclusions: This trial showed that the intranasally administered V. odorata L. oily extract enhances tear production and improves tear film stability., (© 2019 Optometry Australia.)

Different Viola species are known for their traditional use as analgesic, antitussive, febrifuge, hipnotic, analgesic and anti-inflammatory medicinal agents. Additionally, they are considered edible flowers in certain cultures. Thus, the aim of this work was to characterize the phenolic composition and to assess the neuroprotective properties of Viola cornuta and Viola x wittrockiana using in vitro and in vivo methodologies with Caenorhabditis elegans as model. The identification of the phenolic compounds was carried out with a LC-DAD-ESI/MSn. The antioxidant activity of the extracts was determined in vitro using Folin- Ciocalteu, DPPH and FRAP assays and in vivo with a juglone-induced oxidative stress in C. elegans. The neuroprotective properties were evaluated measuring the ability to inhibit CNS enzymes (MAO A, AChE), and the capability to avoid paralyzing the C. elegans CL4176, an Alzheimer disease model. The phenolic content was higher in V. x wittrockiana, being quercetin-3-O-(6-O-rhamnosylglucoside)-7-O-rhamnoside the predominant compound in the extract, which also exhibited a stronger antioxidant capacity in vitro and a higher response to lethal oxidative stress on C. elegans than V. cornuta. Only V. x wittrockiana showed inhibitory effect on CNS enzymes, such as acetylcholinesterase and monoamine oxidase A, but both had protective effect against the paralysis of C. elegans. These findings suggest that the studied V. cornuta and V. x wittrockiana could be interesting candidates for age related neurodegenerative disorder associated with oxidative stress., (Copyright © 2019. Published by Elsevier Taiwan LLC.)

The emergence of rapidly evolving multidrug-resistant pathogens and a deficit of new compounds entering the clinical pipeline necessitate the exploration of alternative sources of antimicrobial therapeutics. Cyclotides revealed in Viola spp . are a class of highly stable, cyclic, and disulfide-bound peptides with diverse intrinsic bioactivities. Herein we have identified a novel complement of 42 putative cyclotide masses in the plant species Viola inconspicua . Cyclotide-containing fractions of a V. inconspicua peptide library revealed potent bioactivities against the Gram-negative bacteria Escherichia coli ATCC 25922 and the highly virulent and multidrug-resistant Klebsiella pneumoniae VK148. As such, six previously uncharacterized cyclotides, cycloviolacins I1-6 (cyI1-cyI6), were prioritized for molecular characterization. Cyclotides cyI3-cyI6 contain a novel "TLNGNPGA" motif in the highly variable loop six region, expanding the already substantial sequence diversity of this peptide class. Library fractions comprised of cyclotides cyI3-cyI6 exhibited MIC values of 18 and 35 μM against E. coli and K. pneumoniae , respectively, whereas isolated cyI3 killed ∼50% of E. coli at 60 μM and isolated cyI4 demonstrated no killing at concentrations >60 μM against both pathogens. This work expands the repertoire of bioactive cyclotides found in Viola spp . and highlights the potential of these antibacterial cyclic peptides.

Viola betonicifolia (Violaceae) is commonly recognized as "Banafsha" and widely distributed throughout the globe. This plant is of great interest because of its traditional, pharmacological uses. This review mainly emphases on morphology, nutritional composition, and several therapeutic uses, along with pharmacological properties of different parts of this multipurpose plant. Different vegetative parts of this plant (roots, leaves, petioles, and flowers) contained a good profile of essential micro- and macronutrients and are rich source of fat, protein, carbohydrates, and vitamin C. The plant is well known for its pharmacological properties, e.g., antioxidant, antihelminthic, antidepressant, anti-inflammatory, analgesic, and has been reported in the treatment of various neurological diseases. This plant is of high economic value. The plant has potential role in cosmetic industry. This review suggests that V. betonicifolia is a promising source of pharmaceutical agents. This plant is also of significance as ornamental plant, however further studies needed to explore its phytoconstituents and their pharmacological potential. Furthermore, clinical studies are needed to use this plant for benefits of human beings.

Background: Pruritus is one of the common problems in hemodialysis patients that involves more than 45% of Iranian patients. Its exact pathogenesis is unclear. Meanwhile, violet is an old plant with anti-scratching effects, which is extensively used to treat diseases in the Persian medicine. This study aimed to evaluate the effect of massaging the body with and without violet oil in the treatment of uremic pruritus., Methods: In this study 57 hemodialysis patients who had uremic pruritus were categorized randomly into two groups. The treatment involved massage with and without topical 5 ml violet oil for 7 min during six sessions (two weeks) on the healthy hand which did not have fistula pruritus score questionnaire were completed before and after the intervention., Results: There was no difference between the two groups before the intervention; however, after the intervention, a significant difference was observed within each group and between the groups in terms of skin dryness score, severity, location, and frequency of pruritus, which were recovered significantly in both groups., Conclusion: The results of this study indicated that massage with violet oil is more effective than massage alone and is recommended as a complementary treatment for patients with uremic pruritis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Breast cancer accounts for one-third of cancer cases in women. Doxorubicin (Dox) is one of the chemotherapeutical compounds widely used to treat breast cancer. Chemical drugs have several side effects and their continuous administration leads to drug resistance in patients. To decrease such side effects in cancer treatment, combination therapy as well as application of natural and herbal compounds has been taken into consideration. The aim of this study was to investigate the cytotoxic effect of Viola odorata (Vo) extract on T47-D human breast cancer cells, alone and in combination with Dox., Materials and Methods: The cytotoxic effects of V. odorata and Dox were studied by morphological examination and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flowcytometric analysis was performed to determine the type of cell death. Moreover, scratch healing assay was conducted to investigate antimigration effect of V. odorata., Results: The results of MTT assay showed that V. odorata and Dox-induced cell death in T47-D cells in a dose- and time-dependent manner. Morphological analysis revealed that V. odorata and Dox-induced features of apoptotic cell death in T47-D cells. These results were confirmed by flow cytometry analysis. Scratch healing assay revealed that migration rate was reduced in the V. odorata- treated cells., Conclusions: Our findings suggest that components of V. odorata exert antitumor effects on human breast cancer and could be administered with lower doses of antitumor agent Dox, in combination therapy, to decrease its side effects., Competing Interests: None

Grouper iridovirus (GIV) is one of the most serious pathogens in mariculture and causes high mortality rates in cultured groupers; then, effective medicines for controlling GIV infections are urgently needed. Viola philippica is a well-known medicinal plant, and the application of V. philippica aqueous extracts against GIV infection was assessed by different methods in this study. The results showed that the working concentration of V. philippica aqueous extracts was 10 mg/ml. V. philippica aqueous extracts below 10 mg/ml have no significant cytotoxic effects on cell viability, while extracts over 15 mg/ml decreased cell viability and showed cytotoxic activity. V. philippica aqueous extracts had excellent inhibitory effects against GIV infection in vitro and in vivo. The possible antiviral mechanism of V. philippica was further analysed, which indicated that V. philippica did no damages to GIV particles, but it could disturb GIV binding, entry and replication in host cells. V. philippica had the best inhibitory effects against GIV during viral infection stage of binding and replication in host cells. Overall, the results suggest that appropriate concentration of V. philippica aqueous extracts has great antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling GIV infection in farmed groupers., (© 2019 John Wiley & Sons Ltd.)

Two lignans including a new one, five flavonoids and five coumarins were isolated from the whole plant of Viola philippica (synonymised as Viola yedoensis Makino). The new compound was structurally determined as (7 R ,8 S ,8' S ) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside by analysis of its NMR, MS and CD spectroscopic data. The known compounds were characterised by comparing their NMR and MS data with those reported. Among the known compounds, 5-hydroxy-4'-methoxyflavone-7-O- rutinoside, 6,7-di-O-β-D- glucopyranosylesculetin, and 7 R ,8 S -dihydrodehydrodiconiferyl alcohol 4-O-β-D- glucopyranoside were isolated and identified from this genus for the first time. Of these compounds, 5-hydroxy-4'-methoxyflavone-7-O-rutinoside and (7 R ,8 S ,8' S ) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside were potently active against α-glucosidase, while the two dimeric coumarins, 5, 5'-bi (6, 7-dihydroxycoumarin) and 6,6',7,7'-tetrahydroxy-5,8'-bicoumarin potently inhibited HCV protease.

Ethanol is the most widely abused drug in the world and its long-term use induces oxidative stress in the liver tissue. The aim of this study was to evaluate protective effect of Viola odorata against ethanol-induced hepatotoxicity in Wistar rat. Animals were divided into 9 groups as follows: control (normal saline), ethanol (10 mg/kg, intraperitoneally), ethanol with 3 doses (125, 250, and 500 mg/kg) of ethyl acetate flower and leaf extracts, and positive control (vitamin E 80 mg/kg). Animals were gavaged 30 min before ethanol injection for 28 days. Then, animals were killed and the livers were separated. Oxidative stress parameters, including reactive oxygen species, lipid peroxidation, and protein carbonyl as well as glutathione content, were evaluated. Also, histopathological examination was performed and assessment of blood alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total antioxidant capacity were evaluated. Ethanol significantly increased oxidative stress markers in liver. Interestingly, administration of both extracts significantly decreased oxidative stress markers in liver tissue and biochemical parameters in the plasma. In addition, abnormal pathological features were improved after treatment with flower and leaf extracts. These results suggested that V. odorata can be considered a candidate for improving conditions due to ethanol-induced tissue oxidative damage because of its antioxidant activity.

Edible flowers consumption and use are an increasing food trend worldwide, although information concerning their nutritional composition and nutraceutical value is still scarce. Thus, the aim of this study was to contribute to the popularization of pansies (Viola × wittrockiana), through the analysis of the nutritional and nutraceutical features of pansies with different colors (white, yellow, and red) and flowering stages. Both flower type and flowering stage influenced the flower composition. When completely open, white and yellow pansies had the highest contents of protein (>2.00 g/100 g fresh weight), while red pansies had the highest content of carbohydrates (8.0 g/100 g fresh weight). Regarding the fatty acid profiles, linoleic acid was always predominant (ranging between 18.7 and 51.0 g/100 g fatty acids), followed by the palmitic and linolenic acids. During flowering, there was an increase in protein, fat, and linolenic acid contents in white and yellow pansies, whereas in red pansies the values did not change. Red pansies were characterized by the highest contents of total carotenoids (873 to 1300 µg β-carotene/g dry weight) and monomeric anthocyanins (303 to 402 µg Cy-3 glu/g dry weight); however, white and yellow pansies showed an increase in the values of total reducing capacity (total phenols), hydrolysable tannins, flavonoids, monomeric anthocyanins, and antioxidant activity from the bud to completely open flower stage. Our results underline the nutritional differences between pansies with different colors at distinct stages of development and their potential health benefits, suggesting that they can be used as ingredient to improve the nutritional properties of foods. PRACTICAL APPLICATION: The market of edible flowers is increasing, although little information in nutritional view is available. So, the present study was conducted to contribute to the popularization of edible flowers as a new and prospective source for the food industry, as well as a promising product for human nutrition. The results of the present study underline the nutritional differences between pansies with different colors at distinct stages of development and their potential health benefits, suggesting that they can be used as ingredient to improve the nutritional properties of foods., (© 2019 Institute of Food Technologists®.)

The color expression of anthocyanin pigments in blue flowers is precisely controlled by their chemical and physical properties such as pH and the presence of metal ions or colorless copigments. Despite the large number of known blue flowers, their coloration mechanisms have not been examined in sufficient detail. In this work, the blue coloration of Viola cornuta petals was expressed via the copigmentation of various flavonol 3- O-glycosides. By using a combination of imaging mass spectrometry with liquid chromatography mass spectrometry, the structures and contents of flavonols colocalized with violanin in the discrete blue-colored regions of the petal were identified. The obtained data allowed the in vitro reconstruction of the color expression that was consistent with the visible spectrum of the viola petal. The results of visible spectral analysis indicated that neither the increase in the solution pH inside the vacuole cells nor the presence of metal ions affected the color development process. Ultimately, it was experimentally confirmed that the excess amounts of flavonol 3- O-glycosides complexed with violanin, which prevented violanin molecules from forming a levorotatory helical self-assembly during the blue color expression via copigmentation.

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Anti-microbial activity and phytochemical analysis of samples from the roots of Viola pilosa was studied against six strains of bacteria. Data indicated that the tested bacterial strain differed in its sensitivity to the root extracts of Viola pilosa. The results showed that Xanthomonas campestris, Bacillus subtilis and S. aureus were more susceptible to butanol extracted fraction. S. aureus was totally resistant to aqueous extracted fraction at all concentrations and .Xanthomonas campestris and Bacillus subtilis were least susceptible to the same fraction at 0.5 mg disc -1 . Similarly, ethyl acetate at 2 mg disc -1 concentrations was effective against Pseudomonas aeruginosa and Escherichia coli. Klebsiella pneumoniae was completely resistant to all the tested concentrations. Phytochemical analysis of the different solvent extracted samples suggested the presence or absence of different various metabolites including alkaloids, saponins, tannins, sterols, flavonoids, protein, carbohydrates and fats.

Widespread resistance to antimicrobial and cancer therapeutics is evolving in every country worldwide and has a direct impact on global health, agriculture and the economy. The specificity and selectivity of bioactive peptide natural products present a possible stopgap measure to address the ongoing deficit of new therapeutic compounds. PepSAVI-MS (Statistically-guided bioActive Peptides prioritized VIa Mass Spectrometry) is an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. This pipeline was validated via screening of the cyclotide-rich botanical species Viola odorata and identification of the known antimicrobial and anticancer cyclotide cycloviolacin O2. Herein we present and validate novel bioactivities of the anthelmintic V. odorata cyclotide, cycloviolacin O8 (cyO8), including micromolar anticancer activity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines and antifungal activity against the agricultural pathogen Fusarium graminearum. A reduction/alkylation strategy in tandem with PepSAVI-MS analysis also revealed several previously uncharacterized putatively bioactive cyclotides. Downstream implementation of ultraviolet photodissociation (UVPD) tandem mass spectrometry is demonstrated for cyO8 as a method to address traditionally difficult-to-sequence cyclotide species. This work emphasizes the therapeutic and agricultural potential of natural product bioactive peptides and the necessity of developing robust analytical tools to deconvolute nature's complexity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Plant-derived natural constituents are of great interest in modern drug discovery due to their natural diversity. Viola odorata L has been traditionally used for the treatment of neuropsychiatric disorders. The present study was undertaken to isolate phytoconstituents including three flavonoids 5,7-Dihydroxy-3,6-dimethoxyflavone[1] 5,7,4'-trihydroxy-3',5'dimethoxyflavone [2] and 5,7,4'-trihydroxy-3'-methoxyflavone [3] from the whole plant of Viola odorata L and to investigate the antidepressant-like effects of these compounds and their possible mechanism of action using antagonists of the serotonergic, dopaminergic and adrenergic system. Classical animal models of depression including tail suspension test (TST) and forced swimming test (FST) using mice were used to evaluate the antidepressant-like effects. Mice were divided into various groups and were administered with either vehicle control, fluoxetine (FLX), or test compounds 1-3 intraperitoneally (i.p.). For experiments involving mechanism determination, mice were pretreated with 5-HT, dopamine and adrenergic antagonists. The brain 5-HT levels were determined following FST. Molecular docking studies were carried out to determine the binding affinity of compounds 1-3 to serotonergic receptors. The results indicated that compounds 1-3 at the dose of 1-30 mg/kg, i.p significantly decreased the immobility time in the FST and TST in mice. The reduction in immobility time was reversed by pre-treating the mice with pCPA (5-HT synthesis inhibitor) 100 mg/kg, i.p. and 5-HT receptor antagonists including WAY100635 (5-HT1a antagonist), ketanserin (a 5-HT2a antagonist) and ondansetron (5-HT3 antagonist) but not with prazosin (α1-adrenergic antagonist) and SCH23390 (D1 dopaminergic antagonist) or haloperidol (D2 dopaminergic antagonist). Moreover, in neurochemical assays, compounds 1-3 caused a significant increase in the 5-HT level in the brain tissue as compared to vehicle. These increases were reversed in the mice groups pretreated with pCPA. Furthermore, molecular docking results also depict that compounds 1-3 can interact with 5HT1A, 5HT2A, and 5HT3 receptors, and are more specific to the 5HT3 receptor subtype. In conclusion, the findings of this study clearly suggest that compounds 1-3 possess antidepressant-like effects which might be mediated via the serotonergic system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The nutritional composition - including total and individual phenolic compounds, carotenoids, and the antioxidant capacity - of two commercially available edible flowers, pansy and snapdragon, was studied. The edible flowers did not differ in their carbohydrates, fat, or ash contents, or in total energy, but pansy had higher values of moisture, protein, and total dietary fiber than snapdragon. Phenolic compounds were more abundant in pansy than in snapdragon, and flavonoids were the major compounds, followed by anthocyanins. The phenolic profile of pansy included flavonols, such as quercetin and isorhamnetin glycosides, flavones, such as apigenin glycosides, and anthocyanins, such as cyanidin and delphinidin glycosides; in snapdragon it included flavonol glycosides (e.g. quercetin and kaempferol glycosides) and anthocyanins, such as cyanidin and pelargonidin glycosides. The contents of total carotenoids were 146 and 29 µg/mg for pansy and snapdragon, respectively, and lutein was the dominant compound., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Violet oil is an ancient herbal drug which has been extensively used to treat insomnia in traditional Iranian Medicine clinics. Violet oil is an almond or sesame oil-based extract of Viola odorata, which is administered as nasal drops. This study aimed to evaluate the efficacy of Violet oil in the treatment of insomnia., Methods and Materials: This study was conducted as a 3-arm double-blind randomized trial. A total of 75 patients with chronic insomnia were enrolled and randomly assigned to three groups in Traditional Iranian Medicine Clinic of Mashhad University of Medical Sciences, Mashhad, Iran. The treatment consisted of intranasal dropping of Violet oil, Almond oil or placebo (1% solution of Carboxymethyl cellulose) in each nostril every night before sleep for 30 days, i.e. three drops of the drug (including either Violet oil or Almond oil) or the placebo was used every night before the sleep. All the patients were asked to complete Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI) questionnaires before and after the intervention., Results: There were no significant differences between patients in the three groups before the intervention (P > 0.05). However, there were significant differences between the three groups after the intervention in ISI scores (P<0.002) and PSQI scores (p<0.001). When comparing the pre- and post-treatment data, the ISI and PSQI scores improved significantly in all the three groups as follows: Violet oil (P<0.001), Almond oil (P<0.001) and placebo (P<0.001). The results also showed that the Violet oil had the most effect among the three groups. In addition, it was more effective on sleep quality than sleep quantity., Conclusion: Considering the effects of natural nasal drug on the improvement of sleep quality in insomniac patients, this study has proposed the use of Violet oil as a natural and herbal drug in a non-oral method without serious side effects for treatment of insomnia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Modification of metal surfaces with antimicrobial peptides is a promising approach to reduce bacterial adhesion. Here, cyclic peptides or cycloids, possessing remarkable stability and antimicrobial activities, were extracted and purified from Viola philippica Cav., and identified using mass spectrometry. Cyclotides were subsequently utilized to modify stainless steel surfaces via polydopamine-mediated coupling. The resulting cyclotide-modified surfaces were characterized by Fourier transform infrared (FTIR) spectroscopy and contact angle analysis. The antibacterial capacity of these cyclotides against Staphylococcus aureus was assessed by Alamar blue assay. The antibiofilm capacity of the modified surfaces was assessed by crystal violet assay, and scanning electron microscopy (SEM). A composite of Kalata b1, Varv A, Viba 15 and Viba 17 (P1); Varv E (P2); and Viphi G (P3) were isolated and identified. FTIR analysis of the modified surfaces demonstrated that cyclotides bound to the surfaces and induced reduction of contact angles. Antimicrobial effects showed an order P3 > P1 and P2, with P3-treated surfaces demonstrating the strongest antibiofilm capacity. SEM confirmed reduced biofilm formation for P3-treated surfaces. This study provides novel evidence for cyclotides as a new class for development of antibacterial and antibiofilm agents., Competing Interests: The authors declare no conflict of interest.

Objectives: Traditional Chinese herbal formulas are difficult to be understood because of complex compositions and specific therapeutic principles. To better understand herbal compatibility in Traditional Chinese medicine (TCM), this study was conducted to investigate the effects of a Chinese pharmacopoeia-listed formula, Erding Formula (EF) and its constituent herbs for a new indication, hyperuricaemia., Methods: A hypoxanthine and potassium oxonate-induced hyperuricemic mouse model, a xylene-induced inflammatory mouse model and an acetic acid-induced pain model were used to test the effects of EF and its constituent herbs. In addition, we investigated whether EF and/or its relevant herbs had an impact on the expression of URAT1 and OAT3 mRNA., Key Findings: The results showed EF and individual herbs had pharmacological effects on selected targets. Only Viola yedoensis Makino (Viola) lowered uric acid levels, while all four herbs had anti-inflammatory and analgesic effects. The EF may lower the uric acid level through inhibiting the expression of URAT1 mRNA and enhancing the expression of OAT3 mRNA., Conclusions: These findings provide pharmacological insights into the effects of EF and individual herbs on UA excretion. This study suggests that Viola is the main herb in EF. This study facilitates better understanding of TCM principles and theories using modern pharmaceutical approaches., (© 2017 Royal Pharmaceutical Society.)

Fifteen alkaloids were isolated from the 95% ethanol extract of the whole plants of Viola yedoensis by various column chromatographic techniques such as silica gel and Sephadex LH-20. Their structures were identified as neoechinulin A（1）,N-benzoyl-L-p-hydroxy-phenylalaninol（2）,aurantiamide acetate（3）,aurantiamide（4）,anabellamide（5）,trichosanatine（6）,indole-3-carboxylic acid methyl ester（7）,3-carboxyindole（8）,N-trans-feruloyl-tyramine（9）,paprazine（10）,7'-（3', 4'-dihydroxyphenyl）-N-[(4-methoxyphenyl)ethyl]propenamide（11）,cannabisin F（12）,N-(4-hydroxyphenethyl)octacosanamide（13）,N-(4-hydroxyphenethyl)hexacosanamide（14）and N-benzoyl-L-phenylalaninol（15）. All the compounds except 3 and 4 were isolated from this plant for the first time. These alkaloids exhibited anti-complement activity against the classical pathway(CP)and the alternative pathway(AP)with the CH50 and AP50 values ranging from 0.12 to 0.33 g•L⁻¹ and 0.22 to 0.50 g•L⁻¹, respectively. Preliminary mechanism study using complement-depleted sera showed that these alkaloids acted on different complement components in the complement activation cascade., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)