Sarracenia alata (Alph.Wood) Alph.Wood Microcuttings as a Source of Volatiles Potentially Responsible for Insects' Respond.

Rare carnivorous plants representing the genus Sarracenia are perceived as very interesting to scientists involved in various fields of botany, ethnobotany, entomology, phytochemistry and others. Such high interest is caused mainly by the unique capacity of Sarracenia spp. to attract insects. Therefore, an attempt to develop a protocol for micropropagation of the Sarracenia alata (Alph.Wood) Alph.Wood, commonly named yellow trumpets, and to identify the specific chemical composition of volatile compounds of this plant in vitro and ex vivo was undertaken. Thus, the chemical volatile compounds excreted by the studied plant to attract insects were recognized with the application of the headspace solid-phase microextraction (HS-SPME) coupled with the GC-MS technique. As the major volatile compounds (Z)-3-hexen-1-ol (16.48% ± 0.31), (E)-3-hexen-1-ol acetate (19.99% ± 0.01) and ß-caryophyllene (11.30% ± 0.27) were identified. Further, both the chemical assumed to be responsible for attracting insects, i.e., pyridine (3.10% ± 0.07), and whole plants were used in in vivo bioassays with two insect species, namely Drosophila hydei and Acyrthosiphon pisum. The obtained results bring a new perspective on the possibilities of cultivating rare carnivorous plants in vitro since they are regarded as a valuable source of bioactive volatile compounds, as including ones with repellent or attractant activity.

Anti-herpes virus activity of the carnivorous botanical, Sarracenia purpurea.

Herpes simplex virus type-1 (HSV-1), one of the most widely spread human viruses in the Herpesviridae family, causes herpes labialis (cold sores) and keratitis (inflammation of the cornea). Conventional treatment for HSV-1 infection includes pharmaceutical drugs, such as acyclovir and docosonal, which are efficacious but maintain the potential for the development of viral drug resistance. Extracts from the carnivorous pitcher plant, Sarracenia purpurea, have previously been shown to inhibit the replication of HSV-1. In this study, we demonstrate that S. purpurea extracts can inhibit the replication of HSV-1 by two distinct mechanisms of action. These extracts directly inhibit extracellular virions or viral attachment to the human host cell as well as inhibiting the expression of viral immediate-early, early and late genes when added at various times post-infection. This botanical has previously been shown to inhibit the replication of poxviruses through the inhibition of early viral gene transcription. These results support a broader anti-viral activity of S. purpurea extracts against both pox and herpes viruses.

Laser Direct Structuring of Bioinspired Spine with Backward Microbarbs and Hierarchical Microchannels for Ultrafast Water Transport and Efficient Fog Harvesting.

Achieving effective dropwise capture and ultrafast water transport is essential for fog harvesting. In nature, cactus uses the conical spine with microbarbs to effectively capture fog, while Sarracenia utilizes the trichome with hierarchical microchannels to quickly transport water. Herein, we combined their advantages to present a novel configuration, a spine with barbs and hierarchical channels (SBHC), for simultaneous ultrafast water transport and high-efficient fog harvesting. This bioinspired SBHC exhibited the fastest water transport ability and the highest fog harvesting efficiency in comparison with the spine with hierarchical channels (SHCs), the spine with barbs and grooves (SBG), and the spine with barbs (SB). Based on the fundamental SBHC unit, we further designed and fabricated a two-dimensional (2D) spider-web-like fog collector and a three-dimensional (3D) cactus-like fog collector using direct laser structuring and origami techniques. The 2D spider-web and 3D cactus-like fog collectors showed high-efficient fog collection capacity. We envision that this fundamental understanding and rational design strategy can be applied in fog harvesting, heat transfer, liquid manipulation, and microfluidics.

Flower scent of Ceropegia stenantha: electrophysiological activity and synthesis of novel components.

In specialized pollination systems, floral scents are crucial for flower-pollinator communication, but key volatiles that attract pollinators are unknown for most systems. Deceptive Ceropegia trap flowers are famous for their elaborate mechanisms to trap flies. Recent studies revealed species-specific floral chemistry suggesting highly specialized mimicry strategies. However, volatiles involved in fly attraction were until now identified in C. dolichophylla and C. sandersonii, only. We here present data on C. stenantha for which flower scent and pollinators were recently described, but volatiles involved in flower-fly communication stayed unknown. We performed electrophysiological measurements with scatopsid fly pollinators (Coboldia fuscipes) and identified 12 out of 13 biologically active floral components. Among these volatiles some were never described from any organism but C. stenantha. We synthesized these components, tested them on antennae of male and female flies, and confirmed their biological activity. Overall, our data show that half of the volatiles emitted from C. stenantha flowers are perceived by male and female fly pollinators and are potentially important for flower-fly communication in this pollination system. Further studies are needed to clarify the role of the electrophysiologically active components in the life of scatopsid fly pollinators, and to fully understand the pollination strategy of C. stenantha.

Nepenthes pitchers are CO2-enriched cavities, emit CO2 to attract preys.

Carnivorous plants of the genus Nepenthes supplement their nutrient deficiency by capturing arthropods or by mutualistic interactions, through their leaf-evolved biological traps (pitchers). Though there are numerous studies on these traps, mostly on their prey capture mechanisms, the gas composition inside them remains unknown. Here we show that, Nepenthes unopened pitchers are CO2-enriched 'cavities', when open they emit CO2, and the CO2 gradient around open pitchers acts as a cue attracting preys towards them. CO2 contents in near mature, unopened Nepenthes pitchers were in the range 2500-5000 ppm. Gas collected from inside open N. khasiana pitchers showed CO2 at 476.75 ± 59.83 ppm. CO2-enriched air-streaming through N. khasiana pitchers (at 619.83 ± 4.53 ppm) attracted (captured) substantially higher number of aerial preys compared to air-streamed pitchers (CO2 at 412.76 ± 4.51 ppm). High levels of CO2 dissolved in acidic Nepenthes pitcher fluids were also detected. We demonstrate respiration as the source of elevated CO2 within Nepenthes pitchers. Most unique features of Nepenthes pitchers, viz., high growth rate, enhanced carbohydrate levels, declined protein levels, low photosynthetic capacity, high respiration rate and evolved stomata, are influenced by the CO2-enriched environment within them.

Metabolite profiling of the carnivorous pitcher plants Darlingtonia and Sarracenia.

Sarraceniaceae is a New World carnivorous plant family comprising three genera: Darlingtonia, Heliamphora, and Sarracenia. The plants occur in nutrient-poor environments and have developed insectivorous capability in order to supplement their nutrient uptake. Sarracenia flava contains the alkaloid coniine, otherwise only found in Conium maculatum, in which its biosynthesis has been studied, and several Aloe species. Its ecological role and biosynthetic origin in S. flava is speculative. The aim of the current research was to investigate the occurrence of coniine in Sarracenia and Darlingtonia and to identify common constituents of both genera, unique compounds for individual variants and floral scent chemicals. In this comprehensive metabolic profiling study, we looked for compound patterns that are associated with the taxonomy of Sarracenia species. In total, 57 different Sarracenia and D. californica accessions were used for metabolite content screening by gas chromatography-mass spectrometry. The resulting high-dimensional data were studied using a data mining approach. The two genera are characterized by a large number of metabolites and huge chemical diversity between different species. By applying feature selection for clustering and by integrating new biochemical data with existing phylogenetic data, we were able to demonstrate that the chemical composition of the species can be explained by their known classification. Although transcriptome analysis did not reveal a candidate gene for coniine biosynthesis, the use of a sensitive selected ion monitoring method enabled the detection of coniine in eight Sarracenia species, showing that it is more widespread in this genus than previously believed.

Antimycobacterial triterpenes from the Canadian medicinal plant Sarracenia purpurea.

ETHNOPHARMACOLOGICAL RELEVANCE: The purple pitcher plant, Sarracenia purpurea, is a medicinal plant used by the Canadian First Nations to treat a wide variety of illnesses. The Mi'kmaq and Wolastoqiyik (Maliseet) peoples of Eastern Canada have traditionally used infusions of S. purpurea for the treatment of tuberculosis-like symptoms. Previous investigations have shown methanolic extracts of S. purpurea to possess antimycobacterial activity. AIM OF THE STUDY: To isolate and identify antimycobacterial constituents from S. purpurea. MATERIALS AND METHODS: Methanolic extracts of S. purpurea were subjected to bioassay guided fractionation using the microplate resazurin assay (MRA) to assess inhibitory activity against Mycobacterium tuberculosis strain H37Ra. The antimycobacterial constituents were identified by NMR, MS and polarimetry. RESULTS: The triterpenes betulinaldehyde, ß-sitosterol, betulinic acid, and ursolic acid were isolated from S. purpurea. Betulinaldehyde, betulinic acid, and ursolic acid exhibited MICs of 450, 950, and 450µM and IC50s of 98, 169, and 93µM against M. tuberculosis H37Ra respectively whilst ß-sitosterol was inactive (MIC and IC50 of >1000µM). CONCLUSIONS: Betulinaldehyde, betulinic acid, and ursolic acid were identified as the principal constituents responsible for the antimycobacterial activity of S. purpurea. This work is consistent with the ethnopharmacological use of S. purpurea by Canadian First Nations as a treatment against infectious diseases.

Phytochemical Comparison of the Water and Ethanol Leaf Extracts of the Cree medicinal plant, Sarracenia purpurea L. (Sarraceniaceae).

PURPOSE: The Cree of Eeyou Istchee in Northern Quebec identified Sarracenia purpurea L. as an important plant for the treatment of Type 2 diabetes. Traditionally the plant is used as a decoction (boiling water extract) of the leaf, however, in order to study the extract in a laboratory setting, an 80% ethanol extract was used. In this study, the phytochemistry of both extracts of the leaves was compared and quantified. METHODS: Two S. purpurea leaf extracts were prepared, one a traditional hot water extract and the other an 80% ethanol extract. Using UPLC-ESI-MS, the extracts were phytochemically compared for 2 triterpenes, betulinic acid and ursolic acid, using one gradient method and for 10 additional substances, including the actives quercetin-3-O-galactoside and morroniside, using a different method. RESULTS: The concentrations of the nine phenolic substances present, as well as an active principle, the iridoid glycoside morroniside, were very similar between the two extracts, with generally slightly higher concentrations of phenolics in the ethanol extract as expected. However, two triterpenes, betulinic acid and ursolic acid, were 107 and 93 times more concentrated, respectively, in the ethanol extract compared to the water extract. CONCLUSION: The main phytochemical markers and most importantly the antidiabetic active principles, quercetin-3-O-galactoside and morroniside, were present in similar amounts in the two extracts, which predicts similar bioactivity.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Evaluation by microarray of the potential safety of Sarracenia purpurea L. (Sarraceniaceae) a traditional medicine used by the Cree of Eeyou Istchee.

PURPOSE: The purpose of this study was to assess safety of the traditional antidiabetic extracts of either S. purpurea or its lead active principle, morroniside at the transcriptional level. The overarching objective was to profile and validate transcriptional changes in the cytochrome P450 family of genes, in response to treatment with S. purpurea ethanolic extract or its lead active, morroniside. METHODS: Transcriptional activity was profiled using a 19K human cDNA microarray in C2BBe1 cells, clone of Caco-2 intestinal cells, which are a model of first-pass metabolism (1, 2). Cells were treated with S. purpurea extract for 4 and 24 hrs, as well as the pure compound morroniside for 4 hrs, to determine their effects. RESULTS: No evidence of cytochrome P450 transcriptome regulation or of transcriptional activation of other diabetes relevant mRNA was detected after rigorous quantitative-PCR validation of microarray results. CONCLUSION: Our data do not support a transcriptional mechanism of action for either S. purpurea extract or its lead active, morroniside. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Slippery liquid-infused porous surface bio-inspired by pitcher plant for marine anti-biofouling application.

Marine biofouling, caused by the adhesion of microorganism, is a worldwide problem in marine systems. In this research work, slippery liquid-infused porous surface (SLIPS), inspired by Nepenthes pitcher plant, was constructed over aluminum for marine anti-biofouling application. The as-fabricated SLIPS was characterized with SEM, AFM, and contact angle meter. Its anti-biofouling performance was evaluated with settlement experiment of a typical marine biofouling organism Chlorella vulgaris in both static and dynamic conditions. The effect of solid substrate micro-structure on anti-biofouling property of SLIPS was studied. It was suggested that the micro-structure with low length scale and high degree of regularity should be considered for designing stable SLIPS with exceptional anti-biofouling property. The liquid-like property is proven to be the main contributor for the exceptional anti-biofouling performance of SLIPS in both static and dynamic conditions. The low roughness, which facilitates removing the settled C. vulgaris under shear force, is also a main contributor for the anti-biofouling performance of SLIPS in dynamic condition.

Photodynamic activity of plant extracts from Sarawak, Borneo.

Photodynamic therapy (PDT) is a medical treatment that involves the irradiation of an administered photosensitizing drug with light of a particular wavelength to activate the photosensitizer to kill abnormal cells. To date, only a small number of photosensitizers have been clinically approved for PDT, and researchers continue to look for new molecules that have more desirable properties for clinical applications. Natural products have long been important sources of pharmaceuticals, and there is a great potential for discovery of novel chemotypes from under-explored biodiversities in the world. The objective of this study is to mine the terrestrial plants in Sarawak, Borneo Island, for new photosensitizers for PDT. In a screening program from 2004 to 2008, we prepared and studied 2,400 extracts from 888 plants for their photosensitizing activities. This report details the bioprospecting process, preparation and testing of extracts, analysis of the active samples, fractionation of four samples, and isolation and characterization of photosensitizers.

Phytochemical constituents of Sarracenia purpurea L. (pitcher plant).

From the leaves of Sarracenia purpurea, collected in Mistissini, Quebec, Canada, four goodyerosides and three phenolics and nine known compounds, were isolated. The structures of the compounds were determined by mass spectrometry, including HRMS, and by 1D and 2D NMR spectroscopy.

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth.

BACKGROUND AND AIMS: Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated. METHODS: Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma-optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes. KEY RESULTS: The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes. CONCLUSIONS: The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.

Characterizing the cytoprotective activity of Sarracenia purpurea L., a medicinal plant that inhibits glucotoxicity in PC12 cells.

BACKGROUND: The purple pitcher plant, Sarracenia purpurea L., is a widely distributed species in North America with a history of use as both a marketed pain therapy and a traditional medicine in many aboriginal communities. Among the Cree of Eeyou Istchee in northern Québec, the plant is employed to treat symptoms of diabetes and the leaf extract demonstrates multiple anti-diabetic activities including cytoprotection in an in vitro model of diabetic neuropathy. The current study aimed to further investigate this activity by identifying the plant parts and secondary metabolites that contribute to these cytoprotective effects. METHODS: Ethanolic extracts of S. purpurea leaves and roots were separately administered to PC12 cells exposed to glucose toxicity with subsequent assessment by two cell viability assays. Assay-guided fractionation of the active extract and fractions was then conducted to identify active principles. Using high pressure liquid chromatography together with mass spectrometry, the presence of identified actives in both leaf and root extracts were determined. RESULTS: The leaf extract, but not that of the root, prevented glucose-mediated cell loss in a concentration-dependent manner. Several fractions elicited protective effects, indicative of multiple active metabolites, and, following subfractionation of the polar fraction, hyperoside (quercetin-3-O-galactoside) and morroniside were isolated as active constituents. Phytochemical analysis confirmed the presence of hyperoside in the leaf but not root extract and, although morroniside was detected in both organs, its concentration was seven times higher in the leaf. CONCLUSION: Our results not only support further study into the therapeutic potential and safety of S. purpurea as an alternative and complementary treatment for diabetic complications associated with glucose toxicity but also identify active principles that can be used for purposes of standardization and quality control.

Antidiabetic compounds from Sarracenia purpurea used traditionally by the Eeyou Istchee Cree First Nation.

Through ethnobotanical surveys, the CIHR Team in Aboriginal Antidiabetic Medicines identified 17 boreal forest plants stemming from the pharmacopeia of the Cree First Nations of Eeyou Istchee (James Bay region of Northern Quebec) that were used traditionally against diabetes symptoms. The leaves of Sarracenia purpurea (pitcher plant), one of the identified Cree plants, exhibited marked antidiabetic activity in vitro by stimulating glucose uptake in C2C12 mouse muscle cells and by reducing glucose production in H4IIE rat liver cells. Fractionation guided by glucose uptake in C2C12 cells resulted in the isolation of 11 compounds from this plant extract, including a new phenolic glycoside, flavonoid glycosides, and iridoids. Compounds 6 (isorhamnetin-3-O-glucoside), 8 [kaempferol-3-O-(6″-caffeoylglucoside], and 11 (quercetin-3-O-galactoside) potentiated glucose uptake in vitro, which suggests they represent active principles of S. purpurea (EC(50) values of 18.5, 13.8, and 60.5 µM, respectively). This is the first report of potentiation of glucose uptake by compounds 6 and 8, while compound 11 (isolated from Vaccinium vitis) was previously shown to enhance glucose uptake. Treatment of H4IIE liver cells with the new compound 1, 6'-O-caffeoylgoodyeroside, decreased hepatic glucose production by reducing glucose-6-phosphatase enzymatic activity (IC(50) = 13.6 µM), which would contribute to lowering glycemia and to the antidiabetic potential of S. purpurea.

In vitro characterization of a nineteenth-century therapy for smallpox.

In the nineteenth century, smallpox ravaged through the United States and Canada. At this time, a botanical preparation, derived from the carnivorous plant Sarracenia purpurea, was proclaimed as being a successful therapy for smallpox infections. The work described characterizes the antipoxvirus activity associated with this botanical extract against vaccinia virus, monkeypox virus and variola virus, the causative agent of smallpox. Our work demonstrates the in vitro characterization of Sarracenia purpurea as the first effective inhibitor of poxvirus replication at the level of early viral transcription. With the renewed threat of poxvirus-related infections, our results indicate Sarracenia purpurea may act as another defensive measure against Orthopoxvirus infections.

Phenolic metabolites in carnivorous plants: Inter-specific comparison and physiological studies.

Despite intensive phytochemical research, data related to the accumulation of phenols in carnivorous plants include mainly qualitative reports. We have quantified phenolic metabolites in three species: Drosera capensis, Dionaea muscipula and Nepenthes anamensis in the "leaf" (assimilatory part) and the "trap" (digestive part). For comparison, commercial green tea was analysed. Phenylalanine ammonia-lyase (PAL) activities in Dionaea and Nepenthes were higher in the trap than in the leaf while the opposite was found in Drosera. Soluble phenols and majority of phenolic acids were mainly accumulated in the trap among species. Flavonoids were abundant in Drosera and Dionaea traps but not in Nepenthes. Phenolic acids were preferentially accumulated in a glycosidically-bound form and gallic acid was the main metabolite. Green tea contained more soluble phenols and phenolic acids but less quercetin. In vitro experiments with Drosera spathulata revealed that nitrogen deficiency enhances PAL activity, accumulation of phenols and sugars while PAL inhibitor (2-aminoindane-2-phosphonic acid) depleted phenols and some amino acids (but free phenylalanine and sugars were elevated). Possible explanations in physiological, biochemical and ecological context are discussed.

A novel resource-service mutualism between bats and pitcher plants.

Mutualistic relationships between vertebrates and plants apart from the pollen and seed-dispersal syndromes are rare. At first view, carnivorous pitcher plants of the genus Nepenthes seem to be highly unlikely candidates for mutualistic interactions with animals, as they form dimorphic terrestrial and aerial pitchers that trap arthropods and small vertebrates. Surprisingly, however, the aerial pitchers of Nepenthes rafflesiana variety elongata are poor insect traps, with low amounts of insect-attractive volatile compounds and low amounts of digestive fluid. Here, we show that N. rafflesiana elongata gains an estimated 33.8 per cent of the total foliar nitrogen from the faeces of Hardwicke's woolly bats (Kerivoula hardwickii hardwickii) that exclusively roost in its aerial pitchers. This is the first case in which the faeces-trapping syndrome has been documented in a pitcher plant that attracts bats and only the second case of a mutualistic association between a carnivorous plant and a mammal to date.

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata.

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.