Kroata Hazler Pilepić

Thin-layer chromatographic analysis of flavonoids, phenolic acids, and amino acids in some Croatian Hypericum taxa

The flowering tops of Hypericum hirsutum, H. montanum, H. perforatum subsp. angustifolium, H. perforatum subsp. perforatum, and H. perforatum subsp. veronense, collected from different locations in Croatia, were investigated to determine their flavonoid, phenolic acid, and amino acid composition. Thin-layer chromatography (TLC) on silica gel was used for separation and identification of the flavonoids and phenolic acids and TLC on cellulose was used for investigation of the amino acids. By use of these procedures ten flavonoids, three phenolic acids, and sixteen amino acids were detected and identified in the samples investigated. The composition of these compounds depended on the taxa investigated and the locality. Spectrophotometric analysis of the flavonoid content indicated that the three subspecies of H. perforatum are richer in flavonoids than H. hirsutum and H. montanum. H. perforatum subsp. perforatum is the richest in flavonoids, phenolic acids, and amino acids. This is the first time these biologically active compounds have been identified in the some Hypericum taxa.

Estimation of phylogenetic relationships among some Hypericum (Hypericaceae) species using internal transcribed spacer sequences

Abstract Thirty-four taxa representing 12 sections of the genus Hypericum were analyzed along with two representatives of Thornea using nuclear internal transcribed spacer (ITS) sequences in order to assess the scope of phylogenetic relationships in this species-rich genus. The resulting parsimony analysis corroborates the actual classification and interspecific affinities. The molecular data provide support for the monophyletic assemblage of the sections Androsaemum, Hypericum, Olympia, and Drosocarpium and for the polyphyletic origin of Ascyreia, Taeniocarpium, Myriandra, and Adenosepalum. The earlier evolutionary hypotheses concerning the origin of Hypericum balearicum and Hypericum calycinum, and the relatedness of Adenosepalum representatives, are not supported by our data analysis as well as hybrid origin of Hypericum perforatum. The obtained results encourage the use of ITS sequence analysis in resolving the phylogenetic relationships within the member-rich genus Hypericum.

LC-PDA-ESI-MSn analysis of phenolic and iridoid compounds from Globularia spp.

Plants produce a great number of metabolites with potentially useful biological activities. Species from the genus Globularia (Plantaginaceae) are known as sources of different phenolic and iridoid compounds. Globularia alypum L. is a medicinal plant used as a healing agent in many Mediterranean countries. Similarities in phytochemical composition are often observed for related species. For Globularia spp., such findings were mostly based on identification of several isolated compounds from distinct species. To our knowledge, this is the first study that enables simultaneous comparison of phytochemical profiles from several members of the genus Globularia. Liquid chromatography-photodiode array detection-electrospray ionization-tandem mass spectrometry was used for the analysis of methanolic extracts of aerial parts obtained from four Globularia species (G. alypum, G. punctata, G. cordifolia and G. meridionalis). In total, 85 compounds were identified or tentatively identified based on comparison of their retention time, UV and MSn (up to MS4 ) spectra to those of standard compounds and/or to literature data. Among these, high relative amounts of bioactive molecules such as globularin, globularifolin, asperuloside and verbascoside (acteoside) were found. Apart from providing new insights into the phytochemistry and chemotaxonomy of selected Globularia species, results of this study complement existing MS/MS spectral data and could enable easier mass spectrometric profiling of certain bioactive compounds such as iridoids and phenylethanoids in related plant species, genera and families. Copyright

RFLP analysis of cpDNA in the genus Hypericum

The chloroplast DNA of 43 species including 16 sections from the genus Hypericum was studied by PCR-RFLP analysis. The PCR-amplified products of four cpDNA regions, trnC-trnD, psbC-trnS, trnL-trnF and rbcL were digested with four restriction endonucleases. A high level of interspecific variation was detected while intraspecific diversity was not observed. The resulting parsimony analysis indicated the monophyletic assemblage of the sections Androsaemum, Olympia, Drosocarpium and Trigynobrathys. Monophyly of Hypericum is weakly supported, but close relationships of H. perforatum and H. maculatum are indicated. The members of Ascyreia are weakly resolved, but clustering of H. kouytchense and H. oblongifolium is well supported, however, H. reptans is nested with Olympia. CpDNA profiles and the positions on the parsimony tree indicate that the chloroplast donor among the putative parents of the hybrid species H. ×inodorum is H. androsaemum.

Chemical composition of the essential oil from Stachys serotina

Stachys serotina (Host) Fritsch is an aromatic, erect, perennial plant similar to Stachys officinalis (L.) Trev. but distinguished clearly in very interrupted inflorescence and later flowering period. The aerial parts of S. serotina were collected on Adriatic island Krk in July 2010. The essential oils were isolated by hydrodistillation for 2 hours. The analyses of the obtained essential oil were performed by GC-FID and GC-MS. Results revealed several differences between Stahys serotina and other Stachys species in Croatia, indicating the existence of a chemical polymorphism. Sesquiterpene hydrocarbons were the most abundant class of isolated volatiles with a predominance of β-caryophyllene (22.6%) followed by δ-cadinene (9.6%), α-humulene (7.5%), germacrene D (6.0%) and minor percentages of others. Determined percentage of β-caryophyllene in Stahys serotina was the highest among other Stachys species from Croatia being most abundant in Stachys officinalis (14.6%). In addition, δ- cadinene was also found with largest abundance in comparison with other Stachys species. Monoterpenes were minor oil constituents of Stachys serotina dominated by monoterpene hydrocarbon α-pinene (11.1%).

Evaluation of the cytotoxic activity of Hypericum spp. on human glioblastoma A1235 and breast cancer MDA MB-231 cells

ABSTRACT Cytotoxic activity of 16 Hypericum ethanolic extracts was evaluated by MTT assay on two human cancer cell lines: glioblastoma A1235 and breast cancer MDA MB-231. Morphology and the type of induced cell death were determined using light and fluorescence microscopy. The majority of Hypericum extracts had no significant cytotoxic effect on MDA MB-231 cells. Eight extracts exhibited mild cytotoxic effect on A1235 cells after 24 h incubation, ranging from 8.0% (H. patulum) to 21.7% (H. oblongifolium). After 72 h of treatment, the strongest inhibition of A1235 viability was observed for extracts of H. androsaemum (26.4–43.9%), H. balearicum (25.8–36.3%), H. delphicum (14.8–27.4%) and H. densiflorum (11.2–24.1%). Micro-scopic examination of cells showed apoptosis as the dominant type of cell death. Due to observed high viability of treated cells, we propose that cytotoxic effects of Hypericum extracts could be related to alternations/interruptions in the cell cycle.

Comparative analysis of specialized metabolites and antioxidant capacity in vitro of different natural populations of Globularia spp.

Abstract Total phenolic, flavonoid, condensed tannin and iridoid content, as well as antioxidant capacity in vitro, were determined spectrophotometrically in methanolic extracts of different plant parts of the Mediterranean medicinal plant Globularia alypum L. and three widespread European species of the same genus: G. cordifolia L., G. meridionalis (Podp.) O. Schwarz and G. punctata Lapeyr. In order to consider possible environmental influences on the production of specialized metabolites, each species, except G. alypum, was collected from three different natural populations. Great variations in the amounts of specialized metabolites were observed among different plant parts and species. For example, total phenolic content ranged from 10.13 (G. punctata, flowers) to 44.90 (G. cordifolia, flower stems) mg gallic acid equivalent g−1 dry weight. Moreover, great differences, attributed to location-specific environmental factors, were observed among different populations of the same species. For example, a strong positive correlation was observed among mean monthly temperatures and total phenolic contents in the leaves of studied Globularia spp. (r = 0.75, p = 0.019). However, despite these differences, all species were rich in bioactive substances when compared to G. alypum, especially in their aerial parts. A very good positive correlation was observed between total phenolic content and DPPH radical scavenging capacity (r = 0.86, p < 0.001)/ABTS radical scavenging capacity (r = 0.83, p < 0.001). The results obtained show that G. cordifolia, G. meridionalis and G. punctata are rich in bioactive substances, providing support for their pharmaceutical utilization. Further investigations are needed to verify the possibility of their medicinal use.

Essential Oil Composition of Three Globularia Species.

The chemical composition of the essential oils obtained by hydrodistillation from the aerial parts of Globularia cordifolia L., G. meridionalis (Podp.) O.Schwarz, and G. punctata Lapeyr. was characterized by GC‐FID and GC/MS analyses. Among the 33 identified compounds, the most abundant present in all investigated samples were oct‐1‐en‐3‐ol (2.9–47.0%), 6‐(1,5‐dimethylhex‐4‐enyl)‐3‐methylcyclohex‐2‐enone (8.2–40.9%), and fukinanolid (7.4–31.6%). Multivariate statistical analyses (PCA and HCA) of the hitherto studied Globularia volatile compounds confirmed to some extent the assumed phylogenetic relationships of the Globularia species studied, including the close relationship between the morphologically similar species G. cordifolia and G. meridionalis, but also evidenced several discrepancies in the current classification of Globularia species.

Analysis of aucubin and catalpol content in different plant parts of four Globularia species

Iridoids are plant secondary metabolites that are gaining more scientific interest due to the wide range of their observed biological activities such as neuroprotective, anti-inflammatory, immunomodulatory, hepatoprotective and cardioprotective. The presence and content of aucubin and catalpol, two iridoid glucosides frequently present in iridoid-containing plants, was studied in methanolic extracts of leaves, flowers, woody stems and underground parts of four Globularia L. species, including the medicinal plant Globularia alypum L., using a specific and reliable HPLC-DAD-ESI/MS method. Aucubin was found in all four species, while catalpol was found only in G. alypum and G. punctata . Flowers contained the highest amounts of investigated iridoids, with catalpol content reaching 1.6% in G. punctata flowers. Comparing to the medicinal plant G. alypum , related species contained higher amounts of investigated iridoids, which makes them interesting candidates for further biological activity investigations. The online version of this article (doi: 10.5073/JABFQ.2015.088.030) contains supplementary files .