Cyclotides derived from Viola dalatensis Gagnep: A novel approach for enrichment and evaluation of antimicrobial activity.

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.

Methods for extraction, isolation and sequencing of cyclotides and others cyclic peptides with anti-helminthic activities: An overview.

The mortality rate caused by parasitic worms on their hosts is of great concern and studies have been carried out to find molecules to reduce the prevalence, host-parasite interaction, and resistance of parasites to treatments. Existing drugs on the market are very often toxic and have many side effects, hence the need to find new, more active molecules. It has been demonstrated in several works that medicinal plants constitute a wide range of new molecules that can solve this problem. Several works have already been able to demonstrate that cyclic peptides of plant origin have shown good activity in the fight against different types of helminths. Therefore, this review aims to provide a general overview of the methods and techniques of extraction, isolation, activities and mechanisms of action of cyclotides and other cyclic peptides for application in the treatment of helminthic infections.

High-throughput microfluidic chip with silica gel-C18 channels for cyclotide separation.

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 (1H-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.

Effects of a plant cyclotide on conformational dynamics and destabilization of ß-amyloid fibrils through molecular dynamics simulations.

Aggregation of ß-amyloid (Aß) peptide is one of the hallmarks of Alzheimer's disease (AD) which results in chronic and progressive neurodegeneration of the brain. A recent study by our group have shown the ability of cyclic disulfide-rich peptides ("cyclotides") isolated from a medicinal plant, Clitoria ternatea, to inhibit the aggregation of Aß peptides and reduce oxidative stress caused by reactive oxygen species using in vivo models of transgenic Caenorhabditis elegans. In the present study, through extensive computational docking and multi-ns molecular dynamics (MD) simulation, we evaluated if cyclotides can stably bind to Aß molecules and/or destabilize the Aß fibril by preventing conformational changes from α-helical to ß-sheet rich structures. We demonstrate that cyclotides bind effectively and stably to different forms of Aß structures via hydrogen bonding and hydrophobic interactions. One of the conserved hydrophobic interface residues, Tyr10 was mutated to Ala and the impact of this virtual mutation was estimated by additional MD simulations for the wild-type (WT) and mutant protein-peptide complexes. A detailed MD simulation analyses revealed that cyclotides form hydrogen bonds with the toxic amyloid assemblies thereby weakening the inter-strand hydrogen bonds between the Aß peptide. The φ-ѱ distribution map of residues in the cyclotide binding pocket that ideally adopt ß-sheet conformation show deviation towards right-handed ɑ-helical (ɑR) conformation. This effect was similar to that observed for the Tyr10Ala mutant and doubly so, for the cyclotide bound form. It is therefore possible to hypothesise that the opening up of amyloid ß-sheet is due to an unfolding process occurring in the Aß caused by cyclotide binding and inhibition. Our current findings provide novel structural insights on the mode of interaction between cyclotides and Aß fibrils and describe their anti-amyloid aggregation potential. This sheds light on the future of cyclotide-based drug design against protein aggregation, a hallmark event in many neurodegenerative diseases.

In vitro Inhibition of HIV-1 by Cyclotide-Enriched Extracts of Viola tricolor.

Since viral infectious diseases continue to be a global health threat, new antiviral drugs are urgently needed. A unique class of therapeutic compounds are antimicrobial peptides (AMPs). They can be found in humans, bacteria and plants. Plants express a wide variety of such defense peptides as part of their innate immune system to protect from invading pathogens. Cyclotides are non-classical AMPs that share a similar structure. Their unique topology consists of a circular peptide backbone and disulfide bonds. In previous studies they have been attributed to a wide range of biological activities. To identify novel cyclotides with antiviral activity, we established a library of plant extracts largely consisting of cyclotide-rich species and screened them as inhibitors of HIV-1 infection. Subsequent extraction and fractionation revealed four cyclotide-containing subfractions from Viola tricolor with antiviral activity. These subfractions inhibited HIV-1 infection with IC50 values between 0.6 and 11.2 µg/ml, and selectivity indices of up to 8.1. The identification and characterization of antiviral cyclotides and the determination of the antiviral mechanisms may allow to develop novel agents to combat viral infections. Therefore, cyclotides represent a natural source of bioactive molecules with prospects for development as therapeutics.

Cationic Clitoria ternatea Seed Peptide as a Potential Novel Bioactive Molecule.

BACKGROUND: While several biologics have been reported from different parts of Clitoria ternatea, a herbaceous climber of the family Fabaceae, specific production of cationic peptides other than cyclotides (<3.7 kDa) has barely been investigated, or their bioactive potential been looked into. OBJECTIVE: The study aims to uncover potential bioactivities and characteristics of novel cationic peptides from C. ternatea seeds. METHODS: C. ternatea seed cationic peptide purified by simple and cost-effective procedures was analyzed by electrophoresis and mass spectrometry. Antimicrobial efficacy was evaluated against bacterial and fungal pathogens. Antioxidant potential was quantified by in vitro antioxidant assays. Physicochemical characterization and Tandem mass spectrometry were performed. RESULTS: An 8.5 kDa cationic peptide purified from C. ternatea seeds was active against Candida albicans, Staphylococcus aureus, Aeromonas hydrophila and Escherichia coli at a minimum inhibitory concentration in the range of 8-32 µg/ml. This activity was totally uncompromised at pH 5-8 or after 1 h of heat treatment at 70-80ºC, but was sensitive to protease treatment. Concentration-dependent free-radical scavenging activity and ferric-reducing capacity demonstrated the antioxidant potential of the peptide. Tandem MS analysis of trypsin-digested peptide based on shotgun proteomics detected matching peptide sequences with one or two cysteine residues but had low sequence coverage (≤17%) to known sequences in the C. ternatea protein database. Taken together, the distinct characteristics of this novel 8.5 kDa peptide clearly distinguish it from known cyclotides of C. ternatea. CONCLUSIONS: Insights have been obtained into the functional characteristics of what appears to be a novel cationic peptide from C. ternatea seeds, exhibiting significant antimicrobial and antioxidant activities.

Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis.

Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1-25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.

Cysteine-rich antimicrobial peptides from plants: The future of antimicrobial therapy.

There has been a spurt in the spread of microbial resistance to antibiotics due to indiscriminate use of antimicrobial agents in human medicine, agriculture, and animal husbandry. It has been realized that conventional antibiotic therapy would be less effective in the coming decades and more emphasis should be given for the development of novel antiinfective therapies. Cysteine rich peptides (CRPs) are broad-spectrum antimicrobial agents that modulate the innate immune system of different life forms such as bacteria, protozoans, fungi, plants, insects, and animals. These are also expressed in several plant tissues in response to invasion by pathogens, and play a crucial role in the regulation of plant growth and development. The present work explores the importance of CRPs as potent antimicrobial agents, which can supplement and/or replace the conventional antibiotics. Different plant parts of diverse plant species showed the presence of antimicrobial peptides (AMPs), which had significant structural and functional diversity. The plant-derived AMPs exhibited potent activity toward a range of plant and animal pathogens, protozoans, insects, and even against cancer cells. The cysteine-rich AMPs have opened new avenues for the use of plants as biofactories for the production of antimicrobials and can be considered as promising antimicrobial drugs in biotherapeutics.

Production of bioactive cyclotides in somatic embryos of Viola odorata.

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.

Coupling Plant-Derived Cyclotides to Metal Surfaces: An Antibacterial and Antibiofilm Study.

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.

An exploration of village-level uterotonic practices in Fenerive-Est, Madagascar.

BACKGROUND: Pharmaceutical uterotonics are effective for preventing postpartum hemorrhage and complications related to unsafe abortion. In Madagascar, however, traditional birth attendants (Matrones) commonly administer medicinal teas for uterotonic purposes. Little is known about Matrone practices and how they might coincide with efforts to increase uterotonic coverage. The aims of this study were to: 1) identify indications for presumed uterotonic plant use by Matrones, 2) explore uterotonic practices at the village level, and 3) describe the response of health practitioners to village-level uterotonic practices. METHODS: Twelve in-depth interviews with health practitioners, Matrones and community agents were conducted in local dialect. All interviews were audio-recorded, transcribed, and translated into English for analysis using Atlas.ti. Medicinal plant specimens were also collected and analyzed for the presence of uterotonic peptides. RESULTS: While Matrones reported to offer specific teas for uterotonic purposes, health practitioners discussed providing emergency care for women with complications associated with use of specific teas. Complications included retained placenta, hypertonic uterus, hemorrhage and sepsis. Chemical analysis indicated the presence of cysteine-rich peptides in the Dantoroa/Denturus plant used in some Matrones' teas. CONCLUSIONS: The presence of uterotonic peptides in one plant used by Matrones may indicate that Matrones intend to administer uterotonics for safer childbirth. This finding, combined with practitioner reports of complications related to some medicinal teas, points to a need for availability of an evidence-based uterotonic at the village level, namely, misoprostol pills or oxytocin in the form of uniject.

Ethnobotanical survey of Rinorea dentata (Violaceae) used in South-Western Nigerian ethnomedicine and detection of cyclotides.

ETHNOPHARMACOLOGICAL RELEVANCE: People living in the tropical rain forest of South-Western Nigeria use Rinorea dentata (P. Beauv.) Kuntze (Violaceae) in ethno-veterinary medicine to facilitate parturition. There are no evidence-based pharmacological investigations for the uterotonic activity of this plant. AIMS OF STUDY: (i) Collection of data about the ethnopharmacological uses of R. dentata and evaluation of its uses and applications in health care; (ii) determining potential uterotonic effects in vitro, and (iii) chemical characterization of R. dentata, which is a member of the Violaceae family known to express circular cystine-knot peptides, called cyclotides. MATERIALS AND METHODS: The ethnopharmacological use of R. dentata in settlement camps within the area J4 of Omo forest has been investigated by semi-structured questionnaires and open interviews. Use index analysis has been performed by seven quantitative statistical models. Respondents' claim on the beneficial ethno-veterinary application of the plant to aid parturition has been investigated in vitro by myometrial contractility organ bath assays. The bioactive plant extract was screened by chemical derivatization and mass spectrometry-based peptidomics using reversed-phase HPLC fractionation and MALDI-TOF/TOF analysis. RESULTS: Based on the survey analysis, medicinal preparations of R. dentata have been used for anti-microbial and anti-malaria purpose in humans, and for aiding parturition in farm animals. The latter application was mentioned by one out of six respondents who claimed to use this plant for any medicinal purpose. The plant extract exhibited a weak uterotonic effect using organ bath studies. The plant contains cyclotides and the peptide riden A has been identified by de novo amino acid sequencing using mass spectrometry. CONCLUSION: Few dwellers around the settlement camps of the tropical forest of Omo (Nigeria) use R. dentata for various health problems in traditional veterinary and human medicine. The weak uterotonic effect of the cyclotide-rich extract is in agreement with the low use value index obtained for this plant. Cyclotides have been reported in the genus Rinorea confirming the ubiquitous expression of these stable bioactive plant peptides within the family of Violaceae.

Microwave-assisted extraction of cyclotides from Viola ignobilis.

Cyclotides are an interesting family of circular plant peptides. Their unique three-dimensional structure, comprising a head-to-tail circular backbone chain and three disulfide bonds, confers them stability against thermal, chemical, and enzymatic degradation. Their unique stability under extreme conditions creates an idea about the possibility of using harsh extraction methods such as microwave-assisted extraction (MAE) without affecting their structures. MAE has been introduced as a potent extraction method for extraction of natural compounds, but it is seldom used for peptide and protein extraction. In this work, microwave irradiation was applied to the extraction of cyclotides. The procedure was performed in various steps using a microwave instrument under different conditions. High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) results show stability of cyclotide structures on microwave radiation. The influential parameters, including time, temperature, and the ratio of solvents that are affecting the MAE potency, were optimized. Optimal conditions were obtained at 20 min of irradiation time, 1200 W of system power in 60 °C, and methanol/water at the ratio of 90:10 (v/v) as solvent. The comparison of MAE results with maceration extraction shows that there are similarities between cyclotide sequences and extraction yields.

Novel cyclotides from Hedyotis diffusa induce apoptosis and inhibit proliferation and migration of prostate cancer cells.

BACKGROUND: Hedyotis diffusa is a well-known herb in traditional Chinese Medicine (TCM) which is used to treat various cancers including prostate cancer. Recently, lots of cyclotides possessing anti-cancer activities were found in Hedyotis family plants, suggesting that H.diffusa may also contain these bioactive ingredients. Cyclotides are heat-stable macrocyclic peptides from plants that display a wide range of biological activities. Currently, over 250 cyclotides have been discovered. OBJECTIVE: This study tried to isolate novel cyclotides from H.diffusa and further investigate their anti-cancer activities for the prostate cancer cells in vitro and in vivo. METHODS: The novel cyclotides from H.diffusa were isolated and purified by High-performance liquid chromatography (HPLC), amino acid sequences in their primary structure were confirmed using Edman degradation and gene cloning. Colorimetric cell viability assay (CCK8 assay), wound healing assay and human prostate cancer xenograft were used to analyze their anti-prostate cancer activity in vitro and in vivo. RESULTS: Three novel cyclotides, termed as Diffusa cyclotide 1 to 3 (DC1-3) from the leaves and root of H.diffusa, were isolated firstly based on my knowledge. Using Edman degradation sequencing and gene cloning, we confirmed their amino acid sequence and obtained precursors of these peptides. By CCK8 assay, all present cyclotides showed potent cytotoxicity against all three prostate cancer cell lines, especially for DC3. In migration assay and wound healing assay, DC3 inhibited the cell migration and invasion Of LNCap cells. By model of prostate xenograft, DC3 could significantly inhibit development of the tumor in weight and size compared to the placebo. CONCLUSION: The novel cyclotides extracted from H.Diffusa have anti-cancer effects, and they are potential bioactive ingredients in H.diffusa.

Screening of Rubiaceae and Apocynaceae extracts for mosquito larvicidal potential.

Rubiaceae and Apocynaceae families are well known for the expression of cyclotides having insecticidal properties. Leaves and flowers extracts of plants from the families Rubiaceae (Ixora coccinea) and Apocynaceae (Allamanda violacea) were evaluated for mosquito larvicidal effect against early IVth instars of Aedes aegypti and Anopheles stephensi. Two forms of plant extracts, one untreated and the other treated with heat and proteolytic enzyme were used for assay. After primary assay, the extract showing more than 50% inhibition was further used for quantification purpose. LC50 and LC90 values of all the extracts were found to be reduced with the treated form. Phytochemical analysis of plant extracts was performed. Primary confirmation for the presence of cyclotides was done by Lowry test, thin layer chromatography and haemolytic assay. This novel approach merits use of plant extracts in mosquito control programmes.

A comparative study of extraction methods reveals preferred solvents for cystine knot peptide isolation from Momordica cochinchinensis seeds.

MCoTI-I and MCoTI-II (short for Momordica cochinchinensis Trypsin Inhibitor-I and -II, respectively) are attractive candidates for developing novel intracellular-targeting drugs because both are exceptionally stable and can internalize into cells. These seed-derived cystine knot peptides are examples of how natural product discovery efforts can lead to biomedical applications. However, discovery efforts are sometimes hampered by the limited availability of seed materials, highlighting the need for efficient extraction methods. In this study, we assessed five extraction methods using M. cochinchinensis seeds, a source of well-characterized cystine knot peptides. The most efficient extraction of nine known cystine knot peptides was achieved by a method based on acetonitrile/water/formic acid (25:24:1), followed by methods based on sodium acetate (20 mM, pH 5.0), ammonium bicarbonate (5 mM, pH 8.0), and boiling water. On average, the yields obtained by these four methods were more than 250-fold higher than that obtained using dichloromethane/methanol (1:1) extraction, a previously applied standard method. Extraction using acetonitrile/water/formic acid (25:24:1) yielded the highest number of reconstructed masses within the majority of plant-derived cystine knot peptide mass range but only accounted for around 50% of the total number of masses, indicating that any single method may result in under-sampling. Applying acetonitrile/water/formic acid (25:24:1), boiling water, and ammonium bicarbonate (5 mM, pH 8.0) extractions either successively or discretely significantly increased the sampling number. Overall, acetonitrile/water/formic acid (25:24:1) can facilitate efficient extraction of cystine-knot peptides from M. cochinchinensis seeds but for discovery purposes the use of a combination of extraction methods is recommended where practical.

Immunosuppressive activity of an aqueous Viola tricolor herbal extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Heartsease (Viola tricolor L.), a member of the Violaceae family, has a long history as a medicinal plant and has been documented in the Pharmacopoeia of Europe. Due to its anti-inflammatory properties it is regarded as a traditional remedy against skin diseases, for example for the treatment of scabs, itching, ulcers, eczema or psoriasis, and it is also used in the treatment of inflammation of the lungs and chest such as bronchitis or asthma. Because T-cells play an important role in the pathological process of inflammatory diseases we investigated the effect of an aqueous Viola extract on lymphocyte functions and explored the 'active' principle of the extract using bioactivity-guided fractionation. MATERIAL AND METHODS: An aqueous Viola extract was prepared by C18 solid-phase extraction. Effects on proliferation of activated lymphocytes (using the cell membrane permeable fluorescein dye CFSE), apoptosis and necrosis (using annexin V and propidium iodide staining), interleukin-2 (IL-2) receptor expression (using fluorochrome-conjugated antibodies) and IL-2 cytokine secretion (using an ELISA-based bead array system) were measured by flow cytometry. Influence on lymphocyte polyfunctionality was characterized by Viola extract-induced production of IFN-γ and TNF-α, as well as its influence on lymphocyte degranulation activity. Fractionation and phytochemical analysis of the extract were performed by RP-HPLC and mass spectrometry. RESULTS: The aqueous Viola extract inhibited proliferation of activated lymphocytes by reducing IL-2 cytokine secretion without affecting IL-2 receptor expression. Similarly, effector functions were affected as indicated by the reduction of IFN-γ and TNF-α production; degranulation capacity of activated lymphocytes remained unaffected. Bioassay-guided fractionation and phytochemical analysis of the extract led to identification of circular plant peptides, so called cyclotides, as bioactive components. CONCLUSION: An aqueous Viola extract contains bioactive cyclotides, which inhibit proliferation of activated lymphocytes in an IL-2 dependent manner. The findings provide a rationale for use of herbal Viola preparations in the therapy of disorders related to an overactive immune system. However, further studies to evaluate its clinical potency and potential risks have to be performed.

Cyclotide discovery in Gentianales revisited--identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants.

Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ∼300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.

From ethnopharmacology to drug design.

Nature's diversity is one of the biggest resources of therapeutic lead compounds. Traditionally-used herbal remedies harbor a variety of bioactive compounds providing researchers with starting points for drug development. Ethnopharmacological investigations of uterotonic plant preparations identified a class of circular and disulfide-rich peptides, called cyclotides, to exhibit strong uterine contractions. In humans one of the physiological regulators of the myometrial contractility is the nonapeptide oxytocin acting on its cognate G protein-coupled receptors. They are considered to represent one of the most promising drug targets with ~30% of all currently marketed drugs acting on these transmembrane receptors. Based on observed similarities regarding the activity and structure of plant cyclotides with human oxytocin we analyzed the pharmacological principle of their action and identified the oxytocin and vasopressin 1a receptors as molecular targets of cyclotides from the Rubiaceae plant Oldenlandia affinis. Using a synthetic approach, the sequence of the native cyclotide kalata B7 was used to design oxytocin-like nonapeptides with nanomolar affinity and selectivity for the oxytocin receptor. This provides formal proof for the use of cyclotides as templates in peptide ligand design. At a more general level, mining of naturally-occurring peptides is a promising tool for the identification and the design of novel G protein-coupled receptor ligands.