Corydalis Cava Research Articles

Applicability of a Blood–Brain Barrier Specific Artificial Membrane Permeability Assay at the Early Stage of Natural Product-Based CNS Drug Discovery

While numerous natural products (NPs) possess activity on central nervous system (CNS) targets, there has been no analytical approach to effectively identify compounds with high brain penetration potential in complex mixtures at the early stage of drug discovery. To overcome this issue, the performance of an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB) for natural products and for plant extracts has been validated and characterized. It was found that the PAMPA-BBB assay preserves its predictive power in the case of natural products and provides high phytochemical selectivity, which enables its use as a unique filtering tool in terms of selecting brain-penetrable compounds from plant extracts. Moreover, the present study has demonstrated that simple modifications in the assay design allow the direct use of PAMPA-BBB filtered samples in a dereplication process, as performed by NMR and LC-MS. The applicability of this procedure was demonstrated using extracts prepared from Tanacetum parthenium, Vinca major, Salvia officinalis, and Corydalis cava, representing different types of chemical diversity and complexity. Thus, the proposed protocol represents a potentially valuable strategy in the NP-based CNS drug discovery environment with a high-throughput screening platform.

Vegetation dynamics of beech forests on limestone in central Germany over half a century – effects of climate change, forest management, eutrophication or game browsing?

The aim of this study was to investigate the vegetation dynamics of suboceanic, submontane, mesic beech forests on limestone that are very rich in spring geophytes over half a century, considering changes in abiotic and biotic conditions including global climate change. Vegetation releves sampled in the Gottinger Wald, southern Lower Saxony, Germany, between 1955 and 1960 (n = 25) and in 1968 (n = 10) were re-surveyed in 2009 on quasi-permanent plots. Differences in species composition, species abundance and vegetation structure were compared between inventories using detrended correspondence analysis (DCA), multiple response permutation procedure (MRPP) and the Wilcoxon signed ranks test. Forty to fifty years ago, a shrub layer was scarcely present, but is common today as a consequence of intensified hunting. An increasing abundance of Allium ursinum, Urtica dioica and Sambucus nigra and a general shift to species composition with higher Ellenberg indicator values for nitrogen can be ascribed to atmospheric nitrogen deposition. The spring geophytes A. ursinum, Corydalis cava and Leucojum vernum can also benefit from global climate change with an earlier start of the vegetation period. A shift towards more oceanic conditions, with mild winters in the past fifty years, may also have increased the competitive strength of evergreen species that are susceptible to long frost periods (e.g. Hedera helix). The resampling of the beech forest vegetation on limestone revealed that many factors have influenced the dynamics over the past half century. These include eutrophication and a reduction in roe deer browsing, but our results also indicate a possible influence of climate change on community composition. In focussing on a small dataset from a very rich sub-unit of beech forests on limestone, the positive response of the spring geophytes becomes particularly obvious.

Ecophysiology of embryo development and seed germination of the European woodland herbaceous perennial Corydalis cava (L.) Schweigg. & Körte subsp. cava (Fumariaceae)

AbstractIn this study we examined the germination ecology with special reference to the temperature requirements for embryo development and germination of Corydalis cava subsp. cava, under both outdoor and laboratory conditions. Corydalis cava is a spring flowering woodland tuberous geophyte widely distributed across Europe. Germination phenology, including embryo development and radicle and cotyledon emergence, was investigated in a population growing in northern Italy. Immediately after harvest, seeds of C. cava were sown both in the laboratory under simulated seasonal temperatures and naturally. Embryos, undifferentiated at the time of seed dispersal, grew during summer and autumn conditions, culminating in radicle emergence in winter, when temperatures fell to ca 5°C. Cotyledon emergence also occurred at ca 5°C, but first emergence was delayed until late winter and early spring. Laboratory experiments showed that high (summer) followed by medium (autumn) and low temperatures (winter) are needed for physiological dormancy loss, embryo development and germination respectively. Unlike seeds of C. cava that germinated in winter, in other Corydalis species radicle emergence occurred in autumn (C. flavula) or did not depend on a period of high summer temperature to break dormancy (C. solida). Our results suggest that subtle differences in dormancy and germination behavior between Corydalis species could be related to differences in their geographical distribution.

Corylucinine, a new Alkaloid from Corydalis cava (Fumariaceae), and its Cholinesterase Activity

A new benzyldihydroisoquinoline alkaloid (1) was isolated from the tubers of Corydalis cava and named corylucinine. Additionally, 8-trichloromethyl-7,8-dihydropalmatine (2), an isolation artifact of tetrahydropalmatine, was obtained. The structures were established by spectroscopic (including 2D NMR and optical rotation) and HR-ESI-MS methods. Both compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. In comparison with the used standards, both compounds showed only moderate inhibitory activity against HuAChE (IC50,. HuAChE = 127.6 +/- 5.2 microM for 1, and IC50, HuAChE = 82.9 +/- 3.9 microM for 2) and none against HuBuChE.

Anticholinesterase and free-radical scavenging activity of Corydalis cava alkaloids

The tubers of Corydalis cava Schweigg. & Kort. were extracted with ethanol and the summary alkaloid extract was fractionated in silica gel chromatography column using step gradient elution with hexane, chloroform and ethanol. Repeated column chromatography, preparative TLC and crystallization led to the isolation of fifteen isoquinoline alkaloids. The chemical structures of isolated compounds were determined on the basis of spectroscopic techniques and by comparison with literature data. Isolated alkaloids were tested on ability to inhibit human erythrocyte acetylcholinesterase, serum butyrylcholinesterase (IC50) and for its free-radical scavenging activities (EC50).

Acetylcholinesterase and Butyrylcholinesterase Inhibitory Compounds from Corydalis Cava (Fumariaceae)

Tubers of Corydalis cava were extracted with ethanol and fractionated using n-hexane, chloroform and ethanol. Repeated column chromatography, preparative TLC and crystallization led to the isolation of fifteen isoquinoline alkaloids. The chemical structures of the isolated compounds were determined on the basis of spectroscopic techniques and by comparison with literature data. All isolated compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. (+)-Canadaline inhibited acetylcholinesterase as well as butyrylcholinesterase in a dose-dependent manner with IC50 values of 20.1 +/- 1.1 microM and 85.2 +/- 3.2 microM, respectively. (+)-Canadine, with an IC50 value of 12.4 +/- 0.9 microM, was the most potent inhibitor of acetylcholinesterase, whilst (+/-)-corycavidine and (+)-bulbocapnine were effective inhibitors of butyrylcholinesterase with IC50 values of 46.2 +/- 2.4 microM and 67.0 +/- 2.1 microM, respectively. The other isolated alkaloids were considered inactive (IC50 > 100 microM).

Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

BackgroundCorydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine), belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers.MethodsNucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS).ResultsThe studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml) - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR) proteins.ConclusionsThe cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.

ChemInform Abstract: Structure of Capnoidine, a Phthalide Isoquinoline Alkaloid

I n t r o d u c t i o n . In the course of our studies of natural products we investigated the crystal and molecular structure of capnoidine isolated from Corydalis cava collected from Vojvodinas Danube region. Extraction, isolation, separation and identification of capnoidine were performed as described by Slavik & Slavikova (1961), Preininger, gimanek, GaSi6 & gantavy (1973) and Ga~i6, Popovi6 & Dragutinovi6 (1985). The interest in this work lies in the accurate determination of molecular conformation of the compound which might assist in a better understanding of its biological action, studies of which are in progress. 0108-2701/91 / 102191-03503.00 Experimental. A crystal 0.22 × 0.35 × 0.15 mm was mounted on a CAD-4 diffractometer (University of Berne) using graphite-monochromated Mo Ka radiation. Cell constants were refined by a leastsquares fit for 22 centered reflections with 0 range from 4.2 to 8.5 ° . Intensity data were collected with w-20 scan in the range 20 3.5tr(/) were considered as observed. Data were corrected for Lorentz and polarization effects. The structure was solved using SHELXS86 (Sheldrick, 1986) with all non-H atoms obtained from the E map (R = 0-173). Full-matrix least-squares (on F) isotropic (R = 0.096) and anisotropic refinement with program SHELX76 (Sheldrick, 1976) minimized ~'.w(AF) 2 for 245 parameters with unit weights. Final R=0 .038 , wR=0.038, (A/tr)max = 0007. Positions of H atoms were generated from assumed geometries and their positions were taken into account without refinement in structure-factor calculations with common isotropic temperature factor U = 0.072 (4)/~2. A final © 1991 International Union of Crystallography 2192 C 2 o H I T N O 6 Table 1. Fractional atomic coordinates (× 104) and equivalent isotropic temperature factors ( x 10 3) I U v~= ~( , , + u~+ v~).

R(−)-Canadaline as first secoberbine alkaloid from Corydalis cava

R(−)-Canadaline ( 1) is the first alkaloid from the class of secoberbines that was found in Corydalis cava. It was identified from a historical collection of substances and its genuine origin was proven. Its absolute configuration was determined by circular dichroism and comparison with published data of the S-enantiomer. Until now only the other enantiomer, S(+)-canadaline, was known to occur in Hydrastis canadensis.

Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Corydalis cava Schweigg. & Kort

In the course of screening plants used in Danish folk medicine as memory enhancers, a crude methanolic extract of tubers from Corydalis cava showed significant acetylcholinesterase inhibitory activity in a dose-dependent manner. Activity guided fractionation of the methanolic extract resulted in the isolation of three alkaloids, bulbocapnine ( 1), corydaline ( 2) and corydine ( 3) as active constituents. Bulbocapnine inhibited acetylcholinesterase as well as butyrylcholinesterase in a dose-dependent manner with IC 50 values of 40 ± 2 μM and 83 ± 3 μM, respectively. Corydaline inhibited acetylcholinesterase in a dose-dependent manner with an IC 50 value of 15 ± 3 μM and corydine inhibited butyrylcholinesterase in a dose-dependent manner with an IC 50 value of 52 ± 4 μM. Corydaline was considered inactive against butyrylcholinesterase and corydine against acetylcholinesterase, due to IC 50 > 100 μM.

Screening of plants used in Danish folk medicine to treat memory dysfunction for acetylcholinesterase inhibitory activity

Aqueous and methanolic extracts of 11 plants, used in Danish folk medicine for improvement of memory and cognition, and 3 Corydalis species were tested for acetylcholinesterase inhibitory activity using the Ellman colorimetric method. Significant inhibitory activity in dose-dependent manner was observed for extracts of Corydalis cava, Corydalis intermedia, Corydalis solida ssp. laxa and Corydalis solida ssp. slivenensis. Extracts of Ruta graveolens, Lavandula angustifolia, Rosmarinus officinalis, Petroselinum crispum and Mentha spicata exhibited moderate inhibition of the enzyme, defined as more than 15% at 0.1 mg/ml.

The fate of Corydalis cava elaiosomes within an ant colony of Myrmica rubra: elaiosomes are preferentially fed to larvae

The diaspores of many plant species are adapted to dispersal by ants (myrmecochory). The ants carry the diaspores into their nests where the appendages of the diaspores (elaiosomes) are consumed. Little is known, however, about the fate of diaspores in the colony, i.e., whether elaiosomes are consumed only by the larvae or also by the workers. In this study, the distribution of Corydalis cava (Fumariaceae) elaiosomes was monitored between the larvae and the working caste of laboratory colonies of Myrmica rubra (Formicidae, Myrmicinae). In the first part of the study, 15N labelled elaiosomes were found to be mainly consumed by larvae. To find out whether elaiosomes are a more attractive food source than an artificial, nutritionally optimized diet for ants (Bhatkar diet), a combined 13C and 15N labelling experiment was conducted. Ants were offered 15N labelled elaiosomes for two days (pulse labelling), while being continuously fed with 13C labelled Bhatkar diet over 19 days. Under the given laboratory conditions, elaiosomes proved to be a far more attractive diet for the larvae than Bhatkar diet, contributing 87 ± 2% of the daily nitrogen and 79 ± 3% of daily carbon incorporation of larvae during the first four days of the experiment. The mean elaiosome incorporation met 73 ± 5% of nitrogen and 63 ± 6% of carbon demand of workers during the first four days of the experiment. Generally, incorporation rates in workers (per dry body mass) were lower both for carbon and nitrogen during the experiment – by a factor of 6.8 for nitrogen and by a factor of 6.2 for carbon compared to larvae. On a colony basis, workers received 39% and larvae 61% of the elaiosome nitrogen. The results indicate that elaiosomes are a major food source for growing larvae and thus support the hypothesis that elaiosomes play an important role in the life cycle of temperate ants.

Positive cooperation of protoberberine type 2 alkaloids from Corydalis cava on the GABA(A) binding site.

Protoberberine alkaloids from the rhizomes of Corydalis cava were investigated with regard to their influence on the GABA A receptor using radioreceptor assays. Whereas the protoberberine type 2 alkaloids, isoapocavidine, corydaline, tetrahydropalmatine, scoulerine and isocorypalmine, increased the specific [(3)H]BMC-binding in a range of 21 - 49 %, the protoberberine type 1 alkaloids, palmatine, coptisine, dehydroapocavidine, and dehydrocorydaline, had no influence on the binding behaviour of the GABA A receptor. To confirm the modulatory activity of the protoberberine type 2 alkaloids on living cells, GABA A receptor binding studies were performed by fluorescence correlation spectroscopy (FCS) using hippocampal neurons and the fluorescently labelled ligand, muscimol-Alexa (Mu-Alexa). The incubation of hippocampal neurons with 7.5 nM Mu-Alexa showed a specific binding of 5.25 nM (70 %). The evaluation of the autocorrelation curve revealed two different mobilities of receptor ligand complexes, D bound1 = (2.8 +/- 0.91) microm 2/s for the free lateral mobility and D bound2 = (0.14 +/- 0.05) microm 2/s for the hindered mobility. An incubation of hippocampal neurons with 7.5 nM Mu-Alexa and 7.5 nM scoulerine showed a maximal increase of the specific Mu-Alexa binding of approximately 27 % by selectively modulating the amount of receptor-ligand complexes with a hindered mobility (9 % to 27 %).

Age determination of individuals of Corydalis species and other perennial herbs

In only a few perennial herbs is age determination of individual plants possible. The life cycle of Corydalis intermedia and C. solida is described. Plants have an age in years identical to the number of dead sheaths outside their underground tuber. Various ways to determine age in other species are reviewed: No. of dead layers around corm: Corydalis cava; no. of dead layers around tuber: Corydalis pumila, Drosera erythrorhiza, D. bulbosa; no. of dead remains of leaves/inflorescence stalks to the side of the corm: Philydrella pygmaea; no. of collaterals in corm: Liatris aspera; no. of nodes on rootstock: Trillium erectum; and no. of bands of high and low density of leaf scars on stems: Aeonium species.

A fatal growth pattern and ways suspected of postponing death: corm dynamics in the perennial herb Corydalis cava

The forest herb Corydalis cava is the only known species with a corm where tissue is added annually as a layer on the outside, and where the older central parts rot away, i.e. the corm becomes an expanding sphere. This growth pattern creates energetic problems for ageing plants. However, older corms change their shape in a way that saves biomass. Growth of corm with age was here modelled introducing different corm traits saving mass and thus postponing death. The largest corm in the study patch had a volume of 60 ml. If such a corm had remained a solid sphere, its age would be estimated to be 8.3 years. In reality, 4 year old corms became hollow and later an opening appeared in their basal part. In addition, corms ≥ 7 years became more conical. A conical corm with a volume of 60 ml was estimated to be 15.9 years old. Volume and shape of corm thus made it possible to age individual plants. The age structure of the plants in the study patch was described and discussed in relation to the overall dynamics of the metapopulation. The patch consisted of a few strong age classes (from 5 to 7 years old). Any recruiting juvenile age classes were almost completely missing.

A fatal growth pattern and ways suspected of postponing death: corm dynamics in the perennial herb Corydalis cava

The forest herb Corydalis cava is the only known species with a corm where tissue is added annually as a layer on the outside, and where the older central parts rot away, i.e. the corm becomes an expanding sphere. This growth pattern creates energetic problems for ageing plants. However, older corms change their shape in a way that saves biomass. Growth of corm with age was here modelled introducing different corm traits saving mass and thus postponing death. The largest corm in the study patch had a volume of 60 ml. If such a corm had remained a solid sphere, its age would be estimated to be 8.3 years. In reality, 4 year old corms became hollow and later an opening appeared in their basal part. In addition, corms ≥ 7 years became more conical. A conical corm with a volume of 60 ml was estimated to be 15.9 years old. Volume and shape of corm thus made it possible to age individual plants. The age structure of the plants in the study patch was described and discussed in relation to the overall dynamics of the metapopulation. The patch consisted of a few strong age classes (from 5 to 7 years old). Any recruiting juvenile age classes were almost completely missing.

Scent profiles of flower colour morphs of Corydalis cava (Fumariaceae) in relation to foraging behaviour of bumblebee queens ( Bombus terrestris)

In general, pollinating insects are discriminatory to flower scent and colour. The perennial forest herb Corydalis cava (Fumariaceae) has either red-magenta or white-flowered individuals. We examined if the different flower colour morphs had different scent profile (headspace-technique, Gas Chromatography-Mass Spectrometry), and if pollinating bumblebees discriminated between colours. Twenty-seven compounds, including monoterpenes, benzenoids, phenylpropanoids and nitrogen-containing compounds were present in the scent samples. Scent profile varied a lot between individual plants regardless of flower colour. The foraging choice from red to either red or white was random. The foraging route, white-white, was, however, chosen much more frequently than one should expect by chance alone. However, number of flowers per inflorescence and fecundity did not differ between colour morphs.

The formation of corydaline and related alkaloids in Corydalis cava in vivo and in vitro

The biosynthesis of corydaline and thalictricavine has been investigated by application of [8-3H]-labelled palmatine and berberine to Corydalis cava bulb tissue. In both cases, high incorporation into the respective (14R,13S)corydaline and -thalictricavine was observed. (S)-[14-3H, 8-14C]Scoulerine was also transferred into corydaline with all of the tritium being lost, indicating that a redox reaction had taken place in the transition of the (14S) to the (14R) product. A partly enriched protein fraction catalyzed the reduction of both protoberberine precursors to the 7,8-dihydro intermediate, which was subsequently methylated at C-13 at the expense of S-adenosylmethionine (SAM) and reduced at C-14 by a B-type reaction with NADPH as reductant. The identity of the product was unequivocally established by NMR, MS, and CD measurements. This biosynthetic route is in full agreement with previously published predictions based on in vivo experiments.

Structure of capnoidine, a phthalide isoquinoline alkaloid

I n t r o d u c t i o n . In the course of our studies of natural products we investigated the crystal and molecular structure of capnoidine isolated from Corydalis cava collected from Vojvodinas Danube region. Extraction, isolation, separation and identification of capnoidine were performed as described by Slavik & Slavikova (1961), Preininger, gimanek, GaSi6 & gantavy (1973) and Ga~i6, Popovi6 & Dragutinovi6 (1985). The interest in this work lies in the accurate determination of molecular conformation of the compound which might assist in a better understanding of its biological action, studies of which are in progress. 0108-2701/91 / 102191-03503.00 Experimental. A crystal 0.22 × 0.35 × 0.15 mm was mounted on a CAD-4 diffractometer (University of Berne) using graphite-monochromated Mo Ka radiation. Cell constants were refined by a leastsquares fit for 22 centered reflections with 0 range from 4.2 to 8.5 ° . Intensity data were collected with w-20 scan in the range 20 3.5tr(/) were considered as observed. Data were corrected for Lorentz and polarization effects. The structure was solved using SHELXS86 (Sheldrick, 1986) with all non-H atoms obtained from the E map (R = 0-173). Full-matrix least-squares (on F) isotropic (R = 0.096) and anisotropic refinement with program SHELX76 (Sheldrick, 1976) minimized ~'.w(AF) 2 for 245 parameters with unit weights. Final R=0 .038 , wR=0.038, (A/tr)max = 0007. Positions of H atoms were generated from assumed geometries and their positions were taken into account without refinement in structure-factor calculations with common isotropic temperature factor U = 0.072 (4)/~2. A final © 1991 International Union of Crystallography 2192 C 2 o H I T N O 6 Table 1. Fractional atomic coordinates (× 104) and equivalent isotropic temperature factors ( x 10 3) I U v~= ~( , , + u~+ v~).

Formation of (R)-configurated tetrahydroprotoberberine alkaloids [formula omitted][formula omitted] and [formula omitted][formula omitted

A highly stereoselective and reversible enzyme system, using NADPH and protoberberine alkaloids, was discovered in Corydalis cava bulbs catalyzing the formation of (R)-configured tetrahydroprotoberberine alkaloids; the hitherto assumed precursor role of (R)-reticuline for these alkaloids is incorrect.