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Determination of huperzine a in Huperzia selago plants from wild population and obtained in in vitro culture by high-performance liquid chromatography using a chaotropic mobile phase

Szypuła W. J, Kiss A. K, Pietrosiuk A, Świst M, Danikiewicz W., Olszowska O

Acta Chromatographica

2011

Vol. 23, no. 2

339--352

This article is the first report describing a new validated method to determine the content of HupA in Huperzia selago (Huperziaceae) from wild population and obtained in in vitro culture using the chaotropic mobile phase. An aqueous-organic (acetonitrile) mobile phase with an added chaotropic salt (NaPF6) was used. The system of mobile phases ensured very high selectivity and efficiency at up to N = 6683 ± 963 theoretical plates calculated for isocratic mode. A Hypercosil GOLD column, C18 250 × 4.6 mm, and a Hypercosil GOLD precolumn, 5UM 10 × 4 mm, were employed for detection at four wavelengths, 230 nm being analytical. The regression coefficient (R2) of the calibration was 0.9993 over the range 25–1252 μg mL -1. The recovery rates were 98.36–105.1% with RSD <2.9%. The intra- and inter-day precisions, expressed as RSD, ranged from 1.2% to 2.7%. LOD for HupA was 14 ng mL -1 for a signal-to-noise ratio of 3:1. The limit of quantification was 140 ng mL. -1. The huperzine A (HupA) content of the plant material ranged from 0.65 mg g−1 dry weight (d.w.) to 1.59 mg g−1 d.w. (material from wild plants) and from 0.44 to 1.10 mg g−1 d.w. (material from in vitro cultures). Interestingly, in our study, plants of H. selago derived from wild population had one of the highest HupA concentrations recorded for a club moss (1.59 mg g -1 d.w.). The findings demonstrate that H. selago, found in Europe and North America, is an alternative source of HupA, richer than H. serrata. In order to confirm that HupA was present in the alkaloid extracts, HPLC-ESI-MS/MS analyses of the patterns were performed in the positive ion mode. The fragmentation quasi-molecular ion of the standard HupA (m/z = 243, [M+H]+) and the ion with m/z = 243 found in the samples were identical, confirming the compound as HupA.

Influence of site conditions on the diversity of endophytic fungi of fir clubmoss Huperzia selago (L.) Bernh. ex Schrank et Mart

Budziszewska J., Szypuła W.

Polish Journal of Ecology

2010

Vol. 58, nr 4

619-626

The aim of the study was to assess the influence of environmental conditions on the composition of endophytic assemblages associated with fir clubmoss, Huperzia selago (L.) Bernh. ex Schrank et Mart. This clubmoss is a cosmopolitan species widely spread in Eurasia and North America, and its gametophytes and sporophytes are colonised by endophytic fungi. Plants for this study were collected in October 2004 and between April and October 2007 from seven European sites, differing in altitude, average annual precipitation, temperature and plant community. Endophytes were isolated on PDA medium (4% potato-dextrose agar) and identified through morphological observations. Different ecological indices were calculated for each site like colonization factor, Fisher's alpha, Shannon's diversity, Morista-Horn and Jaccard's indices. Our results prove that altitude positively correlates with species diversity (Fisher's alpha) and species richness (number of species). Moreover, the similarity assessed from Jaccard's (0.66 for Larici-Piceetum sites) and Morista-Horn indices (0.77 for Juncetea trifidi sites) suggest the influence of plant community on fungal endophyte composition.