Lars Bohlin

Evaluation of anti-inflammatory activity of some Swedish medicinal plants. Inhibition of prostaglandin biosynthesis and PAF-induced exocytosis

Plants used in Swedish traditional medicine to treat inflammatory diseases and/or wounds were selected, based on literature data, for evaluation of inhibitory activity on prostaglandin biosynthesis and platelet activating factor (PAF)-induced exocytosis in vitro. Fifty-nine water extracts from 52 different plants in 28 families were tested. A number of plants, e.g. Calluna vulgaris, Corylus avellana, Geum urbanum, Juniperus communis, Polygonum aviculare, Potentilla erecta and Salix caprea were found to be active in both assays. The most potent cyclooxygenase inhibitors were extracts of Calluna vulgaris, Potentilla erecta and Salix caprea. None of the extracts inhibited just the prostaglandin biosynthesis. In the PAF-test, high inhibition was obtained by 19 extracts, the most potent of which were from Geum rivale, G. urbanum, Solanum dulcamara, Symphytum x uplandicum and Vaccinium vitis-idaea. The in vitro effects in relation to the traditional use, chemical contents and botanical classification, as well as the possibilities and the limitations of the methods are discussed.

Biosynthesis, natural sources, dietary intake, pharmacokinetic properties, and biological activities of hydroxycinnamic acids.

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.

Podophyllotoxin derivatives: drug discovery and development

The exploration and exploitation of podophyllin formulations provide an example of how a plant extract with established ethnomedical use, but also causing toxicity, can provide a basis for new drug discovery and development. Applications and potential applications include the treatment of venereal warts, psoriasis, rheumatoid arthritis, malaria and Alzheimers disease. This review addresses the history and pharmacological action of these natural products and outlines the preclinical development and clinical trials of drugs in the pipeline and with marketing approval.

Cytotoxicity of digitoxin and related cardiac glycosides in human tumor cells.

The saponin digitonin, the aglycone digitoxigenin and five cardiac glycosides were evaluated for cytotoxicity using primary cultures of tumor cells from patients and a human cell line panel (representing different cytotoxic drug-resistance patterns). Of these seven compounds, proscillaridin A was the most potent (IC50: 6.4-76 nM), followed by digitoxin, and then ouabain, digoxin, lanatoside C, digitoxigenin and digitonin. Correlation analysis of the log IC50 values for the cell lines in the panel showed that compound cytotoxicity was only slightly influenced by resistance mechanisms that involved P-glycoprotein, topoisomerase II, multidrug resistance-associated protein and glutathione-mediated drug resistance. Digitoxin and digoxin expressed selective toxicity against solid tumor cells from patients, while proscillaridin A expressed no selective toxicity against either solid or hematological tumor cells. The results revealed marked differences in cytotoxicity between the cardiac glycosides, both in potency and selectivity, and modes of action for cytotoxicity that differ from that of commonly used anticancer drugs.

Recent Insights into the Biosynthesis and Biological Activities of Natural Xanthones

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.

Combined X-ray and NMR Analysis of the Stability of the Cyclotide Cystine Knot Fold That Underpins Its Insecticidal Activity and Potential Use as a Drug Scaffold

Cyclotides are a family of plant defense proteins that are highly resistant to adverse chemical, thermal, and enzymatic treatment. Here, we present the first crystal structure of a cyclotide, varv F, from the European field pansy, Viola arvensis, determined at a resolution of 1.8 Å. The solution state NMR structure was also determined and, combined with measurements of biophysical parameters for several cyclotides, provided an insight into the structural features that account for the remarkable stability of the cyclotide family. The x-ray data confirm the cystine knot topology and the circular backbone, and delineate a conserved network of hydrogen bonds that contribute to the stability of the cyclotide fold. The structural role of a highly conserved Glu residue that has been shown to regulate cyclotide function was also determined, verifying its involvement in a stabilizing hydrogen bond network. We also demonstrate that varv F binds to dodecylphosphocholine micelles, defining the binding orientation and showing that its structure remains unchanged upon binding, further demonstrating that the cyclotide fold is rigid. This study provides a biological insight into the mechanism by which cyclotides maintain their native activity in the unfavorable environment of predator insect guts. It also provides a structural basis for explaining how a cluster of residues important for bioactivity may be involved in self-association interactions in membranes. As well as being important for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptide-based drug design.

Key role of glutamic acid for the cytotoxic activity of the cyclotide cycloviolacin O2.

Abstract.Cyclotides are cyclic plant proteins with potent cytotoxic effects. Here we systematically probed the importance of surface-exposed charged amino acid residues of the cyclotide cycloviolacin O2, using a strategy involving chemical modifications. We show that the single glutamic acid plays a key role for the cytotoxicity: methylation of this residue produced a 48-fold decrease in potency. Virtually no change in potency was observed when masking the single arginine residue using 1,2-cyclohexanedione, while acetylation of the two lysine residues reduced the potency 3-fold. The derivative with modifications at both arginine and lysine residues showed a 7-fold loss of potency. In addition, we show that the activity is dependent on an intact disulfide network and that the short sequences between the six cysteine residues, that is, the backbone loops, are devoid of cytotoxic activity.

Novel Strategies for Isolation and Characterization of Cyclotides: The Discovery of Bioactive Macrocyclic Plant Polypeptides in the Violaceae

This review focuses on the discovery of cyclotides in the Violaceae, their isolation and their anti-cancer effects. These macrocyclic plant peptides consist of about 30 amino acids, including three conserved disulfide bonds in a cystine knotted arrangement, which renders them a remarkable stability. Their unique structure, combined with a wide array of biological activities, makes them of great interest as possible leads in drug development or as carriers of grafted peptide sequences. Here we describe the work conducted in our laboratory, which started with the overall aim of identifying peptides and small proteins of the size 10-50 amino acid residues in plants with novel chemical structures and biological profiles with a potential for drug development or for use as pharmacological tools. Thus we developed a fractionation protocol to directly address major challenges encountered when dealing with plant material, such as removal of chlorophyll, polyphenols, and low molecular compounds omnipresent in plants. Using this protocol, we then discovered a suite of cyclotides, the varv peptides, from the plant Viola arvensis (Violaceae). Following this, separation methods directly targeting cyclotides were developed, e.g. by adsorption, ion exchange chromatography and solvent-solvent partitioning, which then were used in the isolation of additional cyclotides. To structurally examine cyclotides we have also developed methods based on mass spectrometry for cyclotide sequencing and mapping of disulfide bonds. Finally, to assess structure-activity relationships, regarding their anti-cancer and cytotoxic effects that we focus upon, we have also characterized the three dimensional structure of cyclotides by homology modeling techniques.

Biological activity of saponins and saponin-like compounds from starfish and brittle-stars

Twenty-four saponins and saponin-like compounds, isolated from starfish and brittle-stars, have been tested in four in vitro tests, based upon bacterial and cell tissue cultures. Saponin-like compounds from brittle-stars have previously not been tested for biological activity. In an antibacterial test based on an agar diffusion test, the Gram positive bacterium S. aureus was affected by the polyhydroxylated steroidal glycosides, polyhydroxylated sterols and disulfated sterols. However, none of the 21 compounds tested were active against the Gram negative bacterium E. coli. In a cytotoxicity test all 21 compounds tested influenced the cells at a concentration of 100 micrograms/ml, while the cells were unaffected at 1 microgram/ml. In an antitumor test, 16 compounds were tested on two lymphoma cell lines. Inhibition of cell growth, at a concentration of 5 ng/ml, was seen for three polyhydroxylated sterols, in one cell line. Weak activity was seen in an antiviral test at a concentration of 10 micrograms/ml.

Unconventional natural sources for future drug discovery

‘There are more things between heaven and earth…’ Despite the progress of science during the past four centuries, Shakespeares words did not lose their actuality. Knowledge about the etiology of diseases is still limited, and for many life-threatening illnesses no effective treatments exist. Nature always has been a valuable source of drugs and, despite the unprecedented opportunities afforded by medicinal chemistry, continues to deliver lead compounds. Traditionally, research on natural sources was focused on terrestrial plants and microorganisms. More recently, however, organisms of marine origin are also being investigated. Here, the possibilities of unconventional and hardly explored sources are discussed.