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-).
Seven novel macrocyclic polypeptides, designated as varv peptides B-H, have been isolated from the aerial parts of Viola arvensis. Their primary structures have been elucidated by automated Edman degradation and mass spectrometry. They all consist of 29 or 30 amino acid residues, covalently cyclized via the amide backbone and by three internal disulfide bridges. Their amino acid sequences are as follows: varv peptide B, cyclo-(TCFGGTCNTPGCSCDPWPMCSRNGLPVCGE); varv peptide C, cyclo-(TCVGGTCNTPGCSCSWPVCTRNGVPICGE); varv peptide D, cyclo-(TCVGGSCNTPGCSCSWPVCTRNGLPICGE); varv peptide E, cyclo-(TCVGGTCNTPGCSCSWPVCTRNGLPICGE); varv peptide F, cyclo-(TCTLGTCYTAGCSCSWPVCTRNGVPICGE); varv peptide G, cyclo-(TCFGGTCNTPGCSCDPWPVCSRNGVPVCGE); and varv peptide H, cyclo-(TCFGGTCNTPGCSCETWPVCSRNGLPVCGE). The varv peptides B-H exhibited high degrees of homology with the hitherto known macrocyclic peptides varv peptide A, kalata B1, violapeptide I, circulins A and B, and cyclopsychotride A.
The involvement of the glucoalkaloid strictosidine in antimicrobial and antifeedant activity inCatharanthus roseus leaves was investigated. Strictosidine and its deglucosylation product, specifically formed by the enzyme strictosidine glucosidase, were shown to be active against several microorganisms. In contrast, neither the intact glucoside, nor the aglycone product(s) was found to exhibit antifeedant activity againstSpodoptera exigua larvae, as was found for intactC. roseus leaves and leaf extracts. Besides alkaloids further downstream in the biosynthesis pathway, a more apolar, yet unidentified compound may be involved in this activity.
The roots of licorice (Glycyrrhiza glabra) are a rich source of flavonoids, in particular, prenylated flavonoids, such as the isoflavan glabridin and the isoflavene glabrene. Fractionation of an ethyl acetate extract from licorice root by centrifugal partitioning chromatography yielded 51 fractions, which were characterized by liquid chromatography–mass spectrometry and screened for activity in yeast estrogen bioassays. One third of the fractions displayed estrogenic activity towards either one or both estrogen receptors (ERs; ERα and ERβ). Glabrene-rich fractions displayed an estrogenic response, predominantly to the ERα. Surprisingly, glabridin did not exert agonistic activity to both ER subtypes. Several fractions displayed higher responses than the maximum response obtained with the reference compound, the natural hormone 17β-estradiol (E2). The estrogenic activities of all fractions, including this so-called superinduction, were clearly ER-mediated, as the estrogenic response was inhibited by 20–60% by known ER antagonists, and no activity was found in yeast cells that did not express the ERα or ERβ subtype. Prolonged exposure of the yeast to the estrogenic fractions that showed superinduction did, contrary to E2, not result in a decrease of the fluorescent response. Therefore, the superinduction was most likely the result of stabilization of the ER, yeast-enhanced green fluorescent protein, or a combination of both. Most fractions displaying superinduction were rich in flavonoids with single prenylation. Glabridin displayed ERα-selective antagonism, similar to the ERα-selective antagonist RU 58668. Whereas glabridin was able to reduce the estrogenic response of E2 by approximately 80% at 6 × 10−6 M, glabrene-rich fractions only exhibited agonistic responses, preferentially on ERα.Figure Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-5061-9) contains supplementary material, which is available to authorized users.
A multitarget functional bioassay was optimized as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes, mainly to release elastase detected by p-nitroanilide (pNA) formation. Using this bioassay, 100 fractionated extracts of 96 plants were screened, with results presented in a manner that links recorded biological activity to phylogenetic information. The plants were selected to represent a major part of the angiosperms, with emphasis on medicinal plants, Swedish anti-inflammatory plants, and plants known to contain peptides. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. The extract of Digitalis purpurea enhanced pNA formation, and digitoxin, the active compound, was isolated and identified. Plant extracts that exhibited potent nonselective inhibition (>80% inhibition) were evaluated further for direct inhibition of isolated elastase and trypsin enzyme. The inhibitory effect of most tested extracts on the isolated enzyme elastase was similar to that of PAF- and fMLP-induced pNA formation. Compared to trypsin, inhibition of elastase by extracts of Rubus idaeus and Tabernaemontana dichotoma was significantly higher (80% and 99%, respectively). Inhibition of trypsin by the extract of Reseda luteola was high (97%). Orders such as Lamiales and Brassicales were shown to include a comparably high proportion of plants with inhibitory extracts.
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