Renata Jurišić Grubešić

Simultaneous determination of traces of heavy metals by solid-phase spectrophotometry

A coupling sensitive solid phase spectrophotometric (SPS) procedure for determination of traces of heavy metals (Me-SPS) and multicomponent analysis by multiple linear regressions (MA), a simple methodology for simultaneous determination of metals in mixtures was inaugurated. The Me-SPS procedure is based on sorption of heavy metals on PAN-resin and direct absorbance measurements of colour product Me-PAN sorbed on a solid carrier in a 1-mm cell. This methodology (Me-SPS-MA) was checked by simultaneous determination of metals in synthetic mixtures with different compositions and contents of metals important in pharmaceutical practice: Zn, Pb, Cd, Cu, Co, and Ni. Good agreement between experimental and theoretical amounts of heavy metals is obtained from the recovery test (78.3-110.0%). The proposed method enables determination of particular metal ion at the ng mL(-1) level and it was successfully applied to the determination impurities from heavy metal traces in pharmaceutical substances (Cu in ascorbic acid, Pb in glucose, and Zn in insulin). The proposed procedure could be possible contribution to the development of pharmacopoeial methodology for a heavy metals test.

Chemotaxonomic and micromorphological traits of Satureja montana L. and S. subspicata Vis. (Lamiaceae)

Satureja montana and S. subspicata are used as spice, pepper substitute, for preparing tea, juice, and as a medicine. Fourteen populations (seven per species) of Satureja montana L. and S. subspicata Vis. growing in Croatia were examined to determine the chemical composition of the essential oil (analyzed by GC‐FID and GC/MS), the content of macroelements (Na, K, Ca, Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, As) analyzed by ICP‐AES, antioxidant compounds (analyzed by UV/VIS spectrophotometer), and the types and distribution of trichomes (analyzed by scanning electron microscopy). The main constituents of the essential oil were carvacrol and thymol in S. montana (all populations belong to one phenol chemotype), while α‐eudesmol, β‐eudesmol, and spathulenol dominated in S. subspicata (three chemotypes could be distinguished). Both species possess considerably higher quantities of Ca and Mg, and moderate concentrations of K and Na, while Hg and As levels were below the limit of quantification. Non‐glandular trichomes, peltate trichomes, and three types of capitate trichomes were observed on leaves, stem, calyx, and corolla.

Trichome micromorphology in Teucrium (Lamiaceae) species growing in Croatia

Micromorphological investigation of the types, dimensions and distribution of characteristic trichomes in leaves and stems in Teucrium L. species (T. arduini L., T. chamaedrys L., T. flavum L., T. montanum L., T. polium L., and T. scordium L. subsp. scordioides Schreb.) distributed in Croatia was carried out as part of the taxonomical study of the genus Teucrium. Secretory types of hairs, peltate and capitate hairs were observed on the epidermis of stems and leaves of all investigated species. Non-secretory, acicular hairs were almost completely lacking on stems of T. scordium subsp. scordioides. Flagelliform hairs were not found in T. flavum and T. polium. Cladose hairs were present only in T. polium. The largest micromorphological variability was established between wild and cultivated samples of T. arduini and T. scordium subsp. scordioides, while cultivated and wild specimens of T. polium were almost identical. Differences were primarily observed in trichome dimensions and much less in micromorphological features.

Simultaneous RP‐HPLC‐DAD Separation, and Determination of Flavonoids and Phenolic Acids in Plantago L. Species

A rapid reversed‐phase (RP) high‐performance liquid chromatography method was developed and applied for simultaneous separation, and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix, P. coronopus L., P. holosteum Scop. ssp. depauperata Pilger, P. holosteum ssp. holosteum, P. holosteum ssp. scopulorum (Degen) Horvatić, P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB‐C18 using gradient elution with a H2O (pH 2.5, adjusted with CF3COOH) and MeCN mixture at 30°. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum ssp. holosteum), quercetin (max. 0.028%, P. holosteum ssp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was detected only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analyses (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as chemotaxonomic markers in the study of the complex genus Plantago.

Analysis of polyphenols, phytosterols, and bitter principles in Teucrium L. species

Total polyphenols, tannins, β-sitosterol, and bitterness values were determined in native and cultivated populations of Teucrium L. species from Croatia (T. arduini L., T. botrys L., T. chamaedrys L., T. flavum L., T. montanum L., T. polium L., and T. scordium L. subsp. scordioides Schreb.). Teucrium species have long been present in folk medicine for diverse medicinal uses, but little is known about polyphenols, phytosterols, and bitter principles. Spectrophotometrically determined contents of total polyphenols (TP) and tannins (T) significantly varied among investigated Teucrium species and were somewhat higher in native populations. The highest TP and T contents were measured in native specimens of T. montanum (TP: 13.68%; T: 3.48%). Scanning densitometry was used for measurements of β-sitosterol levels in plant samples ranged from 0.056% (T. montanum) to 0.129% (T. botrys), and it was not significantly different between native and cultivated specimens of the same plant species. Bitterness values were similar for native and cultivated samples of the same plant species; the highest was measured for T. montanum (15659). The present study suggests that Teucrium species growing in Croatia have potential for cultivation and might be a valuable source of natural bioactive compounds.

Quantitative analysis of polyphenols and antioxidant activity in four Daphne L. species

The content of biologically active phenolic compounds (total polyphenols, tannins, flavonoids, and phenolic acids) were determined using spectrophotometry in four wild Croatian species of Daphne L. in the family Thymelaeaceae (Daphne alpina, D. cneorum, D. laureola, and D. mezereum). The concentration of total flavonoids (TF) was highest in the leaves of these Daphne species (0.12–0.51% dry herb weight, DW) whereas the content of other phenolic compounds analyzed were highest in the roots, including total polyphenols (TP; 2.71–19.03% DW), tannins (T; 1.14–7.39% DW), and total phenolic acids (TPA; 0.12–0.87% DW). D. alpina contained the highest amount of polyphenols, with the exception of flavonoids, where maximum concentrations were found in D. laureola. We also examined the antioxidant activity of leaf, stem, and root extracts. All extracts analyzed demonstrated high free radical scavenging activity with the highest concentration in the leaf extracts of D. alpina. Leaf extracts of D. cneorum showed the highest antioxidant activity in a β-carotene bleaching assay.

Influence of Soil Traits on Polyphenols Level in Moltkia petraea (Tratt.) Griseb. (Boraginaceae)

Abstract The Illyric–Balkan endemic species Moltkia petraea (Tratt.) Griseb. is very interesting as a potential horticultural and medicinal plant. The aim of this study was to investigate soil conditions of M. petraea habitats, the phenolic content in plant parts, and the influence of soil properties on the phenolic contents. The results were evaluated using Spearman rank order correlations. Analyzed soil samples contained very low to intermediate levels of physiologically active phosphorus, but were very rich in potash. Organic matter content of soil was high. Phenolic compound content was higher in leaves than in flowers or stems. The analyses showed that M. petraea possesses considerable quantities of phenolic compounds and has no specific demands for particular soil conditions. A negative correlation was found between soil phosphorus content and total phenols content in leaves and stems, and with the total phenolic acids content in flowers. Organic matter in soil also found to have a negative influence on total tannins content in stems. Among the tested geographical locations, the Mljet population showed a higher degree of separation from the remaining locations.

Micromorphological differences between some European and American Fraxinus (Oleaceae) species

Micromorphological differences in leaves and pollen between two American (Fraxinus americana L., F. pennsylvanica Marshall) and two European (F. angustifolia Vahl, F. excelsior L.) ash species were studied using scanning electron microscope. The types, dimensions and distribution of characteristic trichomes were established and measured. Capitate hairs on the leaves had the same shape in all researched ash species. Acicular hairs were regularly present in two American ash species, but very rarely in the glabrous phase of F. angustifolia and F. excelsior. Only F. americana had coronulate abaxial surface of leaves. Pollen of F. angustifolia and F. excelsior had 3 (tricolpate) apertures, and F. americana and F. pennsylvanica 4 (stephanocolpate) apertures. Based on the appearance of the reticulum it’s possible to clearly distinguish all four species. F. angustifolia and F. pennsylvanica had muri with transversal ridges and seldom granules. Muri of F. excelsior and F. americana had slightly visible transversal ridges, and because of that noticeable granules.

Fruit and seed traits of Berberis croatica Horvat and Berberis vulgaris L.

Fruit and seed traits of Berberis croatica Horvat and Berberis vulgaris L. The three year variability of fruits and seeds was investigated in Berberis croatica (Vošac, Mt Biokovo, Rakov Potok near Zagreb) and in B. vulgaris (Fran Kušan Pharmaceutical Botanical Garden in Zagreb). Berberis croatica had the following dimensions of fruits (seeds): length 7.28-7.88 (4.57-5.03) mm; width 3.85-3.99 (width 1: 2.06-2.20; width 2: 1.44-1.63) mm; weight 0.065-0.078 (0.0116-0.0134) g. Dimensions of B. vulgaris fruits (seeds) were: length 10.20-11.29 (5.71-6.24) mm; width 5.29-5.83 (width 1: 2.40-2.71; width 2: 1.60-1.98) mm; weight 0.1602-0.2199 (0.0146-0.0235) g. The fruit shape of both species was similar and the length/width ratio was 1.91-2.04 in B. croatica and 1.77-2.07 in B. vulgaris. The number of seeds per fruit was 1.23-1.58 in B. croatica and 1.36-1.54 in B. vulgaris. Generally, fruits and seeds of B. vulgaris were significantly longer, wider and heavier than fruits and seeds of B. croatica. ANOVA showed significant statistical differences between populations for all analyzed fruit and seed traits while the species significantly differed in all traits, except in the fruit shape and number of seeds in fruit.