Naturally occurring cystine knot peptides show a wide range of biological activity, and as they have inherent stability they represent potential scaffolds for peptide-based drug design and biomolecular engineering. Here we report the discovery, sequencing, chemical synthesis, three-dimensional solution structure determination and bioactivity of the first cystine knot peptide from Cactaceae (cactus) family: Ep-AMP1 from Echinopsis pachanoi. The structure of Ep-AMP1 (35 amino acids) conforms to that of the inhibitor cystine knot (or knottin) family but represents a novel diverse sequence; its activity was more than 500 times higher against bacterial than against eukaryotic cells. Rapid bactericidal action and liposome leakage implicate membrane permeabilisation as the mechanism of action. Sequence homology places Ec-AMP1 in the plant C6-type of antimicrobial peptides, but the three dimensional structure is highly similar to that of a spider neurotoxin.
Cyclotides are ultrastable plant proteins characterized by the presence of a cyclic amide backbone and three disulfide bonds that form a cystine knot. Because of their extreme stability, there has been significant interest in developing these molecules as a drug design scaffold. For this potential to be realized, efficient methods for the synthesis and oxidative folding of cyclotides need to be developed, yet we currently have only a basic understanding of the folding mechanism and the factors influencing this process. In this study, we determine the major factors influencing oxidative folding of the different subfamilies of cyclotides. The folding of all the cyclotides examined was heavily influenced by the concentration of redox reagents, with the folding rate and final yield of the native isomer greatly enhanced by high concentrations of oxidized glutathione. Addition of hydrophobic solvents to the buffer also enhanced the folding rates and appeared to alter the folding pathway. Significant deamidation and isoaspartate formation were seen when oxidation conditions were conducive to slow folding. The identification of factors that influence the folding and degradation pathways of cyclotides will facilitate the development of folding screens and optimized conditions for producing cyclotides and grafted analogs as stable peptide-based therapeutics.
The Solanaceae is an important plant family that has been playing an essential role in traditional medicine and human nutrition. Members of the Solanaceae are rich in bioactive metabolites and have been used by different tribes around the world for ages. Antimicrobial peptides (AMPs) from plants have drawn great interest in recent years and raised new hope for developing new antimicrobial agents for meeting the challenges of antibiotic resistance. This review aims to summarize the reported AMPs from plants of the Solanaceae with possible molecular mechanisms of action as well as to correlate their traditional uses with reported antimicrobial actions of the peptides. A systematic literature study was conducted using different databases until August 2019 based on the inclusion and exclusion criteria. According to literature, a variety of AMPs including defensins, protease inhibitor, lectins, thionin-like peptides, vicilin-like peptides, and snaking were isolated from plants of the Solanaceae and were involved in their defense mechanism. These peptides exhibited significant antibacterial, antifungal and antiviral activity against organisms for both plant and human host. Brugmansia, Capsicum, Datura, Nicotiana, Salpichora, Solanum, Petunia, and Withania are the most commonly studied genera for AMPs. Among these genera, Capsicum and the Solanum ranked top according to the total number of studies (35%-38% studies) for different AMPs. The mechanisms of action of the reported AMPs from Solanaceae was not any new rather similar to other reported AMPs including alteration of membrane potential and permeability, membrane pore formation, and cell aggregation. Whereas, induction of cell membrane permiabilization, inhibition of germination and alteration of hyphal growth were reported as mechanisms of antifungal activity. Plants of the Solanaceae have been used traditionally as antimicrobial, insecticidal, and antiinfectious agents, and as poisons. The reported AMPs from the Solanaceae are the products of chemical shields to protect plants from microorganisms and pests which unfold an obvious link with their traditional medicinal use. In summary, it is evident that AMPs from this family possess considerable antimicrobial activity against a wide range of bacterial and fungal pathogens and can be regarded as a potential source for lead molecules to develop new antimicrobial agents.
This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
The structures of the recently published monoterpene indole alkaloids penduflorines A and B (1a and 1b), isolated from Tabernaemontana penduliflora (Apocynaceae), have been revised. Rather than an inseparable mixture of two compounds, they appear to be the known alkaloid vobasine (2). Although we could not comprehensively revise the structures of penduflorines C-E due to lacking spectral data, since their structural elucidations were based on that of 1a and 1b, their structures should also be treated with caution.
This work combines the wound-healing-related properties of the host defense peptide KR-12 with wood-derived cellulose nanofibrils (CNFs) to obtain bioactive materials, foreseen as a promising solution to treat chronic wounds. Amine coupling through carbodiimide chemistry, thiol-ene click chemistry, and Cu(I)-catalyzed azide-alkyne cycloaddition were investigated as methods to covalently immobilize KR-12 derivatives onto CNFs. The effects of different coupling chemistries on the bioactivity of the KR12-CNF conjugates were evaluated by assessing their antibacterial activities against Escherichia coli and Staphylococcus aureus. Potential cytotoxic effects and the capacity of the materials to modulate the inflammatory response of lipopolysaccharide (LPS)-stimulated RAW 245.6 macrophages were also investigated. The results show that KR-12 endowed CNFs with antibacterial activity against E. coli and exhibited anti-inflammatory properties and those conjugated by thiol-ene chemistry were the most bioactive. This finding is attributed to a favorable peptide conformation and accessibility (as shown by molecular dynamics simulations), driven by the selective chemistry and length of the linker in the conjugate. The results represent an advancement in the development of CNF-based materials for chronic wound care. This study provides new insights into the effect of the conjugation chemistry on the bioactivity of immobilized host defense peptides, which we believe to be of great value for the use of host defense peptides as therapeutic agents.
Currents efforts in marine biodiscovery have essentially focused on temperate to tropical shallow water organisms. With more than 6000 species of marine plants and animals, the Kosterfjord area has the richest marine biodiversity in Swedish waters, but it remains understudied. The overall objective of our marine pharmacognosy research is to explore and reveal the pharmacological potential of organisms from this poorly explored region. More generally, we wish to understand aspects of structure-activity relationships of chemical interactions in cold-water marine environment (shallow and deep). Our strategy is based on ecologically guided search for compounds through studies of physiology and organism interactions coupled to identification of bioactive molecules guided by especially in vivo assays. The research programme originated in the beginning of the 1980s with a broad screening of Swedish marine organisms using both in vitro and in vivo assays, resulting in isolation and identification of several different bioactive molecules. Two congenerous cyclopeptides, i.e. barettin and 8,9-dihydrobarettin, were isolated from the deep-sea sponge Geodia barretti, and structurally elucidated, guided by their antifouling activity and their affinity to a selection of human serotonin receptors. To optimize the activity a number of analogues of barettin were synthezised and tested for antifouling activity. Within the EU project BlueGenics, two larger homologous peptides, barrettides A and B, were isolated from G. baretti. Also, metabolic fingerprinting combined with sponge systematics was used to further study deep-sea natural product diversity in the genus Geodia. Finally, the chemical property space model 'ChemGPS-NP' has been developed and used in our research group, enabling a more efficient use of obtained compounds and exploration of possible biological activities and targets. Another approach is the broad application of phylogenetic frameworks, which can be used in prediction of where-in which organisms-to search for novel molecules or better sources of known molecules in marine organisms. In a further perspective, the deeper understanding of evolution and development of life on Earth can also provide answers to why marine organisms produce specific molecules.
With a realistic threat against biodiversity in rain forests and in the sea, a sustainable use of natural products is becoming more and more important. Basic research directed against different organisms in Nature could reveal unexpected insights into fundamental biological mechanisms but also new pharmaceutical or biotechnological possibilities of more immediate use. Many different strategies have been used prospecting the biodiversity of Earth in the search for novel structure-activity relationships, which has resulted in important discoveries in drug development. However, we believe that the development of multidisciplinary incentives will be necessary for a future successful exploration of Nature. With this aim, one way would be a modernization and renewal of a venerable proven interdisciplinary science, Pharmacognosy, which represents an integrated way of studying biological systems. This has been demonstrated based on an explanatory model where the different parts of the model are explained by our ongoing research. Anti-inflammatory natural products have been discovered based on ethnopharmacological observations, marine sponges in cold water have resulted in substances with ecological impact, combinatory strategy of ecology and chemistry has revealed new insights into the biodiversity of fungi, in depth studies of cyclic peptides (cyclotides) has created new possibilities for engineering of bioactive peptides, development of new strategies using phylogeny and chemography has resulted in new possibilities for navigating chemical and biological space, and using bioinformatic tools for understanding of lateral gene transfer could provide potential drug targets. A multidisciplinary subject like Pharmacognosy, one of several scientific disciplines bridging biology and chemistry with medicine, has a strategic position for studies of complex scientific questions based on observations in Nature. Furthermore, natural product research based on intriguing scientific questions in Nature can be of value to increase the attraction for young students in modern life science.
In many countries today, the number of students selecting chemistry for higher studies is decreasing. At the same time, interest in the environmental aspects of chemistry, green chemistry, and sustainable use of natural products is increasing among the young generation of students. By modernizing and renewing a venerable proven science, pharmacognosy would have a strategic position to connect biology and chemistry. This multidisciplinary subject is important for discovery of novel and unique molecules with drug potential, and for revealing unknown targets, by studying evolutionary structure-activity optimization in nature. In this paper, the overall aim and strategies of our research are presented and exemplified by three different research projects.
Natural products are involved in scientific issues important for a sustainable society, and a multidisciplinary subject such as pharmacognosy can, therefore, be useful in increasing future interest in both chemistry and biology.
Ethnopharmacological relevance: Leptopyrum fumarioides has been used in the traditional medicine of Mongolia for the treatment of various diseases, including drug intoxications. However, since there is only sparse information about its chemistry, active components, and pharmacological and toxicological effects, the major aim of the present study employing mouse lymphoma cells was to evaluate the genotoxic and antigenotoxic/antioxidative effects of extracts and components isolated from this plant. Material and methods: A crude methanol extract was separated into three different sub-extracts: dichloromethane, n-butanol, and water. The major constituent of the n-butanol extract, i.e., the flavone luteolin-7-O-glucoside and a mixture of the most abundant compounds in the dichloromethane sub-extract were then isolated. DNA damage was evaluated using the comet assay; the antioxidant activity was evaluated using the DPPH radical scavenging assay. Results: The crude methanol extract, the dichloromethane sub-extract and the mixture of compounds isolated from the latter fraction, increased the level of DNA damage after three hours of exposure. In contrast, no increase in DNA damage was observed in the cells that had been exposed to the n-butanol and water sub-extracts, or to the pure flavone. When non-DNA damaging concentrations of extracts and compounds were tested together with the DNA damaging agent catechol, all sub-extracts were found to reduce the catechol-induced DNA damage (the flavone was then found to be the most effective protective agent). The n-butanol sub-extract and the flavone were also found to have the most prominent antioxidative effects. Conclusion: Based on the results from the present study, components in Leptopyrum fumarioides were found to protect the DNA damage induced by catechol, probably by acting as potent antioxidants.
The Ricinus communis biomarker peptides RCB-1 to -3 comprise homologous sequences of 19 (RCB-1) or 18 (RCB-2 and -3) amino acid residues. They all include four cysteine moieties, which form two disulfide bonds. However, neither the 3D structure nor the biological activity of any of these peptides is known. The synthesis of RCB-1, using microwave-assisted, Fmoc-based solid-phase peptide synthesis, and a method for its oxidative folding are reported. The tertiary structure of RCB-1, subsequently established using solution-state NMR, reveals a twisted loop fold with antiparallel ?-sheets reinforced by the two disulfide bonds. Moreover, RCB-1 was tested for antibacterial, antifungal, and cytotoxic activity, as well as in a serum stability assay, in which it proved to be remarkably stable.
Hypa A, a novel macrocyclic polypeptide containing 30 amino acid residues, has been isolated from the n-butanol extract of the Argentine plant Hybanthus parviflorus. The sequence, cyclo-(SCVYIPCTITALLGCSCKNKVCYNGIPCAE), was determined by automated Edman degradation, quantitative amino acid analysis and nanospray MS/MS(2). Three intramolecular disulfide bridges stabilize the cyclic peptide backbone of hypa A. Using these structural features to classify the peptide as a cyclotide, we extended the distribution of that substance class to a new genus, and now propose a uniform nomenclature for cyclotides.
Hypa A, a novel macrocyclic polypeptide containing 30 amino acid residues, has been isolated from the n-butanol extract of the Argentine plant Hybanthus parviflorus. The sequence, cyclo-(SCVYIPCTITALLGCSCKNKVCYNGIPCAE), was determined by automated Edman degradation, quantitative amino acid analysis and nanospray MS/MS2. Three intramolecular disulfide bridges stabilize the cyclic peptide backbone of hypa A. Using these structural features to classify the peptide as a cyclotide, we extended the distribution of that substance class to a new genus, and now propose a uniform nomenclature for cyclotides.
Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.
Cyclotides stand out as the largest family of circular proteins of plant origin hitherto known, with more than 280 sequences isolated at peptide level and many more predicted from gene sequences. Their unusual stability resulting from the signature cyclic cystine knot (CCK) motif has triggered a broad interest in these molecules for potential therapeutic and agricultural applications. Since the time of the first cyclotide discovery, our laboratory in Uppsala has been engaged in cyclotide discovery as well as the development of protocols to isolate and characterize these seamless peptides. We have also developed methods to chemically synthesize cyclotides by Fmoc-SPPS, which are useful in protein grafting applications. In this review, experience in cyclotide research over two decades and the recent literature related to their structures, synthesis, and folding as well the recent proof-of-concept findings on their use as "epitope" stabilizing scaffolds are summarized.
The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability and active in a wide range of biological activities make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (Möbius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity and derivatives in which the Trp residue was modified showed low effect, suggesting the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the Möbius cyclotides. However, despite these differences, a systematic test of mixtures of cyclotides, even from both subfamilies revealed that they act in an additive way.
During last decade there has been increased interest in the small, cysteine-rich cyclotide proteins found in plant species of the violet family (Violaceae). These cyclotides consist of a circular chain of approximately 30 amino acids, including six cysteines that form three disulfide bonds, arranged in a cyclic cystine knot motif. In this study we map the occurrence and distribution of cyclotides in the Violaceae. Plant material was obtained from herbarium sheets containing samples up to 200 years old. Even the oldest specimens exhibited a remarkable stability of cyclotides in the preserved leaves, with no degradation products observable, making them one of the most stable proteins in nature. We analyzed the cyclotide content in over 200 samples covering 17 of the 23 genera, and positively identified cyclotides in almost 150 of approximately 900 known species in the Violaceae. Each species contained a unique set of between one and 25 cyclotides, of which many were exclusive to individual species. The estimated number of different cyclotides in the Violaceae is 5,000-25,000. We conclude that the Violaceae is an extremely rich source of cyclotides, and we propose that cyclotides are ubiquitous among all Violaceae species.
The cyclotide family of plant-derived peptides is defined by a cyclic backbone and three disulfide bonds locked into a cyclic cystine knot. They display a diverse range of biological activities, many of which have been linked to an ability to target biological membranes. In the current work, we show that membrane binding and disrupting properties of prototypic cyclotides are dependent on lipid composition, using neutral (zwitterionic) membranes with or without cholesterol and/or anionic lipids. Cycloviolacin O2 (cyO2) caused potent membrane disruption, and showed selectivity towards anionic membranes, whereas kalata B1 and kalata B2 cyclotides were significantly less lytic towards all tested model membranes. To investigate the role of the charged amino acids of cyO2 in the membrane selectivity, these were neutralized using chemical modifications. In contrast to previous studies on the cytotoxic and antimicrobial effects of these derivatives, the Glu6 methyl ester of cyO2 was more potent than the native peptide. However, using membranes of Escherichia coil lipids gave the opposite result: the activity of the native peptide increased 50-fold. By using a combination of ellipsometry and LC-MS, we demonstrated that this unusual membrane specificity is due to native cyO2 extracting preferentially phosphatidylethanolamine-lipids from the membrane, i.e., PE-C16:0/cyC17:0 and PE-C16:0/C18:1.
The cyclotides are a family of plant-derived proteins that occur in plants from the Violaceae (violet) and Rubiaceae (coffee) families and have a diverse range of biological activities, including cytotoxic, hemolytic, antimicrobial, and insecticidal activities; the latter suggests their natural function lies in plant defense. In the current study we have investigated the membrane-disrupting and adsorption ability of prototypic cyclotides and correlated these findings to their cytotoxic properties. We also included modifications of selected charged amino acids in cycloviolacin O2, previously shown to be of importance for its cytotoxic activity. The cyclotides’ cytotoxic activity, ability to adsorb and disrupt model lipid membranes of different charge densities was investigated, e.g. by fluorescence spectroscopy, ellipsometry, and circular dichroism. Cytotoxicity of the native cyclotides was demonstrated to correlate to membrane adsorption and lytic activity. Hence, the activity of native cyclotides is mainly due to interactions between the proteins and the phospholipids in the target membrane. Striking effects of single amino acid variations in cycloviolacin O2 on its membrane interaction were also demonstrated.
Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v. 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v. 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC50 values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2–3 days; lower doses were without complications.
During the last decade there has been increasing interest in small circular proteins found in plants of the violet family (Violaceae). These so-called cyclotides consist of a circular chain of approximately 30 amino acids, including six cysteines forming three disulfide bonds, arranged in a cyclic cystine knot (CCK) motif. In this study we map the occurrence and distribution of cyclotides throughout the Violaceae. Plant material was obtained from herbarium sheets containing samples up to 200 years of age. Even the oldest specimens contained cyclotides in the preserved leaves, with no degradation products observable, confirming their place as one of the most stable proteins in nature. Over 200 samples covering 17 of the 23-31 genera in Violaceae were analyzed, and cyclotides were positively identified in 150 species. Each species contained a unique set of between one and 25 cyclotides, with many exclusive to individual plant species. We estimate the number of different cyclotides in the Violaceae to be 5000-25,000, and propose that cyclotides are ubiquitous among all Violaceae species. Twelve new cyclotides from six phylogenetically dispersed genera were sequenced. Furthermore, the first glycosylated derivatives of cyclotides were identified and characterized, further increasing the diversity and complexity of this unique protein family.
Two disulfide-containing peptides, barrettides A (1) and B (2), from the cold-water marine sponge Geodia barretti are described. Those 31 amino acid residue long peptides were sequenced using mass spectrometry methods and structurally characterized using NMR spectroscopy. The structure of 1 was confirmed by total synthesis using the solid-phase peptide synthesis approach that was developed. The two peptides were found to differ only at a single position in their sequence. The three-dimensional structure of 1 revealed that these peptides possess a unique fold consisting of a long beta-hairpin structure that is cross-braced by two disulfide bonds in a ladder-like arrangement. The peptides are amphipathic in nature with the hydrophobic and charged residues clustered on separate faces of the molecule. The barrettides were found not to inhibit the growth of either Escherichia coli or Staphylococcus aureus but displayed antifouling activity against barnacle larvae (Balanus improvisus) without lethal effects in the concentrations tested.
A fractionation protocol for the isolation of a highly purified polypeptide fraction from plant biomass is described. The procedure dereplicates ubiquitous substance classes known to interfere with bioassays often used in natural product-based drug discovery programs. The protocol involves pre-extraction with dichloromethane, extraction with ethanol (50%), removal of tannins with polyamide, removal of low-molecular-weight components with size-exclusion chromatography over Sephadex G-10, and final removal of salts and polysaccharides with solid-phase extraction using reversed-phase cartridges. The method has been applied to the aerial parts of Viola arvensis, resulting in the isolation of a peptide fraction that on further separation yielded a novel 29-residue macrocyclic polypeptide named varv peptide A, cyclo(-TCVGGTCNTPGCSCSWPVCTRNGLPVCGE-).
In 2015, an International Union of Pure and Applied Chemistry (IUPAC) Task Group was formed to develop nomenclature recommendations for homodetic cyclic peptides produced from ribosomal precursors. Delegates of the 2015 International Conference on Circular Proteins (ICCP) were presented with the strengths and weaknesses of four published approaches to homodetic cyclic peptide nomenclature, and a summary of the ensuing discussion is presented here. This interim report presents a potentially novel suggestion-the use of Cahn-Ingold-Prelog rules to specify amino acid priority in homodetic peptides for consistent numbering. Indeed, this might be the first extension of the Cahn-Ingold-Prelog rules in five decades. The authors invite interested parties to contact the corresponding author with suggestions for the improvement of the proposed nomenclature; these ideas will be discussed and considered for inclusion in the final report.
A challenge for the next generation marine antifouling (AF) paints is to deliver minimum amounts of biocides to the environment. The candidate AF compound medetomidine is here shown to be released at very low concentrations, ie ng ml(-1) day(-1). Moreover, the release rate of medetomidine differs substantially depending on the formulation of the paint, while inhibition of barnacle settlement is independent of release to the ambient water, ie the paint with the lowest release rate was the most effective in impeding barnacle colonisation. This highlights the critical role of chemical interactions between biocide, paint carrier and the solid/aqueous interface for release rate and AF performance. The results are discussed in the light of differential affinity states of the biocide, predicting AF activity in terms of a high surface affinity and preserved bioavailability. This may offer a general framework for the design of low-release paint systems using biocides for protection against biofouling on marine surfaces.
An extract of Glinus lotoides, a medicinal plant used in Africa and Asia for various therapeutic purposes, was recently shown to cause DNA damage in vitro. To further explore the potential genotoxicity of this plant, fractionation of the crude extract was performed using reverse phase solid-phase extraction and a stepwise gradient elution of methanol in water. Four fractions were collected and subsequently analysed for their DNA damaging effects in mouse lymphoma cells using an alkaline version of the comet assay. To identify potential genotoxic and non-genotoxic principles, each fraction was then subjected to liquid chromatography coupled to mass spectrometry, LC-MS/MS. 1D and 2D nuclear magnetic resonance analyses were used to confirm the identity of some saponins. Although fractions containing a mixture of flavonoids and oleanane-type saponins or oleanane-type saponins alone produced no DNA damage, those containing hopane-type saponins exhibited a pronounced DNA damaging effect without affecting the viability of the cells. To conclude, even if this study presents evidence that hopane-type of saponins are endowed with a DNA damaging ability, further studies are needed before individual saponins can be cited as a culprit for the previously reported genotoxicity of the crude extract of G. lotoides.
Hydroxycinnamic acids are the most widely distributed phenolic acids in plants. Broadly speaking, they can be defined as compounds derived from cinnamic acid. They are present at high concentrations in many food products, including fruits, vegetables, tea, cocoa, and wine. A diet rich in hydroxycinnamic acids is thought to be associated with beneficial health effects such as a reduced risk of cardiovascular disease. The impact of hydroxycinnamic acids on health depends on their intake and pharmacokinetic properties. This review discusses their chemistry, biosynthesis, natural sources, dietary intake, and pharmacokinetic properties.
Ethnopharmacological relevance: Medicinal plants from the Sinai desert are widely used in traditional Bedouin medicine to treat a range of conditions including, cancers, and may thus be useful sources of novel anti-tumor compounds. Information on plants used in this way was obtained through collaboration with Bedouin herbalists. Aim of the study: To document the traditional uses of 61 species from 29 families of Egyptian medicinal plants and to investigate their biological activity using a cytotoxicity assay. Material and methods: MeOH extracts of the 61 plant species investigated were dissolved in 10% DMSO and their cytotoxic activity was evaluated. The extracts were tested in duplicate on three separate occasions at three different concentrations (1, 10 and 100 mu g/ml) against human lymphoma U-937 GTB. The most active extract was subjected to bioassay-guided fractionation using HPLC and LC/ESI-MS to isolate and identify its active components. Results and discussion: The most potent extracts were those from Asclepias sinaica, Urginea maritima, Nerium oleander and Catharanthus roseus, followed by those from Cichorium endivia, Pulicaria undulate and Melia azedarach. Literature reports indicate that several of these plants produce cardiac glycosides. Bioassay-guided fractionation of alcoholic U. maritima extracts led to the isolation of a bioactive bufadienolide that was subsequently shown to be proscillaridin A, as determined by 1D and 2D NMR spectroscopy. This result demonstrates the value of plants used in traditional medicine as sources of medicinally interesting cytotoxic compounds.
This review focuses on recent advances in our understanding of the complex biosynthetic pathways and diverse biological activities of naturally occurring xanthones. The biosynthesis section covers studies published from 1989 to 2008 on xanthone production in plants and fungi, while the bioactivity review presents tabulated activities of more than 250 xanthones described in studies published from 2001 to 2008, together with structural information and indications of their wide-ranging potential uses as pharmacological tools. A large number of relevant papers have been published on these subjects (128 cited here), illustrating the diversity of the xanthones and their possible uses.
In this review, an updated literature survey covering the reports of naturally occurring xanthones in the period of 2005-2008 is presented. In some 143 studies, the isolation of 264 different xanthones from 36 plant species (representing 15 genera in 6 families of higher plants), 7 species of fungi, and 1 lichen species were reported. Of these, 122 compounds were isolated for the first time from nature. We discuss plant origin, the way of separation, and spectral analysis done for structure elucidation, along with a brief discussion of the chemosystematic significance.
Bioassay-guided isolation using an in vitro assay testing for anti-schistosomiasis yielded a novel triterpene saponin, asparagalin A, from the n-butanol extract of the roots of Asparagus stipularis Forssk., Asparagaceae. The structure was elucidated by spectroscopic analysis and chemical transformations. Administration of asparagalin A resulted in a retardation of worm growth and locomotion at the first day and showed a significant activity of egg-laying suppression at 200 mu g/mL concentration.
In our search for effective tick repellents from plant origin, we investigated the effect of essential oils of four medicinal and culinary plants belonging to the family Lamiaceae on nymphs of the tick Ixodes ricinus (L.). The essential oils of the dry leaves of Rosmarinus officinalis (Rosemary) (L.), Mentha spicata (Spearmint) (L.), Origanum majorana (Majoram) (L.), and Ocimum basilicum (Basil) (L.) were isolated by steam distillation and 15 mu g/cm(2) concentration of oils was tested against ticks in a laboratory bioassay. The oils of R. officinalis, M. spicata, and O. majorana showed strong repellency against the ticks 100, 93.2, and 84.3%, respectively, whereas O. basilicum only showed 64.5% repellency. When tested in the field, the oils of R. officinalis and M. spicata showed 68.3 and 59.4% repellency at a concentration of 6.5 mu g/cm(2) on the test cloths. The oils were analyzed by gas chromatography mass spectrometry and the major compounds from the most repellent oils were 1,8-cineole, camphor, linalool, 4-terpineol, borneol, and carvone.
Introduction: We have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA). Methods: The autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC (R) system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP) 2 using the CCPlus (R) ELISA kit. Results: Two peptides derived from the fibrinogen a chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen beta chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency. Conclusions: Here we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.
Cyclotides, the largest known family of head-to-tail cyclic peptides, have approximately 30 amino acid residues with a complex structure containing a circular peptide backbone and a cystine knot. They are found in plants from the Violaceae and Rubiaceae families and are speculated to function in plant protection. In addition to their insecticidal properties, cyclotides display cytotoxic. anti-IIIV, antimicrobial, and inhibition of neurotensin binding activities. Although cyclotides are present in all violaceous species hitherto screened, their distribution and expression in Rubiaceae are not fully understood. In this study, we show that Psychotria leptothyrsa var. longicarpa (Rubiaceae) contains a suite of different cyclotides. The cyclotide fractions were isolated by RP-HPLC, and sequences of six new peptides, named psyles A-F. were determined by MS/MS sequencing. One or these, psyle C. is the first rubiaceous linear variant known. Psyles A, C, and E were analyzed in a fluorometric microculture assay to determine cytotoxicity toward the human lymphoma cell line U937-GTB. The IC50 values of psylcs A. C. and E were 26, 3.50, and 0.76 mu M. respectively. This study expands the number of known rubiaceous cyclotides and shows that the linear cyclotide maintains cytotoxicity.
Cyclotides are a large family of plant peptides characterized by their cyclic cystine knot composed of a circular backbone and three disulfide bonds that impart exceptional stability. They, and several acyclic variants, have been isolated from plants within the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Poaceae families. A variety of chemical and genetic approaches have been applied for the discovery and characterization of cyclotides. As investigations of cyclotide expression, distribution, and phylogeny rapidly increase, the authors have proposed the inclusion of information pertaining to plant species that have been analyzed but do not appear to express cyclotides into the CyBase database. CyBase is dedicated to providing web tools and information about cyclic peptides and proteins to the scientific community. Including detailed information about sampling and analysis parameters of plant species that have been investigated but not published elsewhere should assist in the process of selecting species for establishing new cyclotide discovery projects, as well as for detailed reanalysis using alternative technical approaches. In summary, the collection and deposition of all plant species that have been examined (whether cyclotides have been found or not) would help to impart a deeper understanding of cyclotide discovery, evolution, and physiological function.
Cycloviolacin O2 (CyO2), a cyclotide from Viola odorata (Violaceae) has antitumor effects and causes cell death by membrane permeabilization. In the breast cancer line, MCF-7 and its drug resistant subline MCF-7/ADR, the cytotoxic effects of CyO2 (0.2-10 mu M) were monitored in the presence and absence of doxorubicin (0.1-5 mu M) using cell proliferation assays to establish its chemosensitizing abilities. SYTOX Green assays were performed to verify membrane permeabilization and showed cellular disruption correlates with cyclo tide chemosensitization. Fluorescence microscopy studies demonstrated increased cellular internalization of doxorubicin in drug resistant cells when coexposed to CyO2. Interestingly, CyO2 did not produce significant membrane disruption in primary human brain endothelial cells, which suggested cyclo tide specificity toward induced pore formation in highly proliferating tumor cells. Furthermore, three novel cyclotides (psyle A, C and E) from Psychotria leptothyrsa (Rubiaceae) were also monitored for cytotoxic activity. The cyclotides displayed potent cytotoxicity (IC50 = 0.64->10 mu M), and coexposure to cyclotides significantly enhanced doxorubicin induced toxicity (IC50 = 0.39-0.76 mu M). This study documents several cyclotides with robust cytotoxicity that may be promising chemosensitizing agents against drug resistant breast cancer.
Human immunodeficiency virus type-1 (HIV-1), the etiologic agent of acquired immune deficiency syndrome (AIDS), is a global pandemic causing millions of deaths annually. Highly active antiretroviral therapy (HAART) greatly enhances lifespan but eventually causes debilitating side effects, in part, due to their chronic administration required to suppress HIV-1 replication. If treatment is discontinued, viral suppression is lost and dormant replication-competent monocytic cell reservoirs become reactivated, leading to viral recrudescence and progression to AIDS. Therefore, novel strategies to circumvent obstacles to HIV-1 therapy are critically needed. We evaluated the potentially therapeutic effects of cycloviolacin O2 (CyO2) on cell viability (MTTassay), membrane disruption (SYTOX Green uptake), p24 production [enzyme-linked immunosorbent assays (ELISA)], and proviral integration (PCR amplification) in U1 cells; a monocytic cell model of HIV-1 latency and reactivation. We demonstrate, for the first time, that CyO2 (0.5-5.0 mu M) kills productively infected cells. Sub-toxic concentrations (< 0.5 mu M) of CyO2 disrupted plasma membranes in both latently-infected and productively-infected U1 cells and enhanced the antiviral efficacy of nelfinavir, a HIV-1 protease inhibitor (HPI). Interestingly, CyO2 also decreased virus production by activated U1 cells; however, this effect was not due to suppression of integrated provirus in U1 cells. This suggested that, in addition to the known pore-forming ability of cyclotides, a novel mode of antiviral activity may exist for CyO2. Our data indicate that CyO2 may be a promising candidate for the targeting HIV-1 reservoirs in monocytes, and their inclusion in adjuvant therapy approaches may augment the efficacy of HPIs and ultimately facilitate virus elimination.