Antifungal bioassay

Crude extracts and pure compounds were tested in 96-well microtiter plate assays against the phytopathogenic ascomycetes Botrytis cinerea Pers. and Septoria tritici Desm. according to the fungicide resistance action committee (FRAC) with the following modifications (FRAC, 2015a, 2015b, 2015c). Briefly, crude extracts were examined at 1250 and 416.7 µ g/ml, while pure compounds were tested at 250, 83.3 and 27.8 µM. The solvent DMSO was used as negative control (max. concentration 2.5%), while the commercial fungicide pyraclostrobin (Sigma Aldrich, Germany) served as positive control (100% inhibition at 83.3 µM). Five to seven days after inoculation, pathogen growth was evaluated by measurement of the optical density (OD) at λ 405 nm with a TecanGENios Pro microplate reader (5 measurements per well using multiple reads in a 3 × 3 square). Each experiment was carried out in triplicates.

135 6.3.6. Antiviral assay

Antiviral activity of pure compounds and crude extract against herpes simplex virus, type 1 (HSV-1, ATCC, VR-1383) was tested in a plaque assay using Vero monolayer in a 96-well plate (Vero cells, ATCC, CCL-81). Cell viability was determined by the addition of PrestoBlue (Invitrogen) and spectrophotometric reading for fluorescence using Softmax Pro (Molecular Devices, Ca). The methods (unpublished) used for this assay are described in detail in the supplemental material.

Appendix. Supporting Information (this is available online)

Supplemental material (antiviral assays, ¹H and ¹³C NMR spectra of compounds 6.1-6.10, ECD spectrum and chiral HPLC chromatogram of compound 6.2) associated with this chapter is available online at http://www.sciencedirect.com/science/article/pii/S0031942216300206.

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Chapter 7

New source report: chemical constituents of Psorospermum densipunctatum (Hypericaceae)

Graphical abstract*

Highlights

• Characterization of three prenylated anthraquinones and five proanthocyanidins from the chemically unexplored P. densipunctatum

• First report on proanthocyanidins from the genus Psorospermum

• Discussion on the chemotaxonomic significance of the isolated compounds

*This part (with slight modifications) was published: Fobofou, S.A.T., Franke, K., Schmidt, J., Wessjohann, L., 2015. Biochem. Syst. Ecol. 59C, 174-176. Reprinted (adapted) with permission from the Copyright Clearance Center (confirmation number: 11472956).

P. densipunctatum

139 Abstract

The present work describes the chemistry of the phytochemically unexplored Psorospermum densipunctatum, which (like Hypericum species) belongs to the family Hypericaceae. Eight natural products, including three prenylated anthraquinones (7.1-7.3) and five proanthocyanidins (7.5-7.8) were isolated from P. psorospermum for the first time. The occurrence of prenyltated compounds in this plant is of high chemotaxonomic significance since they can be markers of the family Hypericaceae. Also, this is the first report of proanthocyanidins (7.5-7.8) from the genus Psorospermum.

Keywords: Prenylated anthraquinones; Proanthocyanidins; Psorospermum densipunctatum;

Hypericaceae.

140