Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities.

Tropaeolum majus L. is a traditional medicinal plant with a wide range of biological activities due to the degradation products of the glucosinolate glucotropaeolin. Therefore, the goals of this study were to identify volatiles using gas chromatography-mass spectrometry analysis (GC-MS) of the hydrosols (HYs) isolated using microwave-assisted extraction (MAE) and microwave hydrodiffusion and gravity (MHG). Cytotoxic activity was tested against a cervical cancer cell line (HeLa), human colon cancer cell line (HCT116), human osteosarcoma cell line (U2OS), and healthy cell line (RPE1). The effect on wound healing was investigated using human keratinocyte cells (HaCaT), while the antibacterial activity of the HYs was tested against growth and adhesion to a polystyrene surface of Staphylococcus aureus and Escherichia coli. Antiphytoviral activity against tobacco mosaic virus (TMV) was determined. The GC-MS analysis showed that the two main compounds in the HYs of T. majus are benzyl isothiocyanate (BITC) and benzyl cyanide (BCN) using the MAE (62.29% BITC and 15.02% BCN) and MHG (17.89% BITC and 65.33% BCN) extraction techniques. The HYs obtained using MAE showed better cytotoxic activity against the tested cancer cell lines (IC50 value of 472.61-637.07 µg/mL) compared to the HYs obtained using MHG (IC50 value of 719.01-1307.03 µg/mL). Both concentrations (5 and 20 µg/mL) of T. majus HYs using MAE showed a mild but statistically non-significant effect in promoting gap closure compared with untreated cells, whereas the T. majus HY isolated using MHG at a concentration of 15 µg/mL showed a statistically significant negative effect on wound healing. The test showed that the MIC concentration was above 0.5 mg/mL for the HY isolated using MAE, and 2 mg/mL for the HY isolated using MHG. The HY isolated using MHG reduced the adhesion of E. coli at a concentration of 2 mg/mL, while it also reduced the adhesion of S. aureus at a concentration of 1 mg/mL. Both hydrosols showed excellent antiphytoviral activity against TMV, achieving100% inhibition of local lesions on the leaves of infected plants, which is the first time such a result was obtained with a hydrosol treatment. Due to the antiphytoviral activity results, hydrosols of T. majus have a promising future for use in agricultural production.

Isolation of Volatile Compounds by Microwave-Assisted Extraction from Six Veronica Species and Testing of Their Antiproliferative and Apoptotic Activities.

This study was conducted to determine the differences in the chemical composition of the essential oils and hydrosols of six different Veronica species (V. agrestis, V. anagalloides, V. austriaca ssp. jacquinii, V. beccabunga, Veronica cymbalaria, and V. officinalis) and to test their antiproliferative and apoptotic activities, according to the authors' knowledge, because of insufficient research and lack of information. Also, the goal was to determine which obtained samples were better in achieving antiproliferative and apoptotic activities and due to which volatile components. Therefore, essential oils (EOs) and hydrosols (HYs) were isolated from the above-mentioned Veronica species by microwave-assisted extraction (MAE). Phytochemical identification of the free volatile compounds was performed using a GC equipped with a flame ionization detector and a mass spectrometer. Their antiproliferative and apoptotic activities against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, were determined. The main compounds identified in the studied Veronica EOs and HYs were terpinen-4-ol (0.34-6.49%), linalool (0.34-6.61%), (E)-caryophyllene (0.97-7.55%), allo-aromadendrene (0.18-2.21%), caryophyllene oxide (1.42-23.83%), benzene acetaldehyde (0.26-13.34%), and ß-ionone (1.08-16.53%). In general, HYs of the tested Veronica species showed higher antiproliferative activity (IC50 13.41-42.05%) compared to EOs (IC50 158.1-970.4 µg/mL) on MDA-MB-231 and T24 cancer cell lines after 48 and 72 h. V. agrestis EO showed the best apoptotic effect among the EOs on the MDA-MB-231 cancer cell line (10.47 ± 0.53% and 9.06 ± 0.74% of early/late apoptosis, compared with control 3.61 ± 0.62% and 0.80 ± 0.17% of early/late apoptosis, respectively) and among the HYs V. cymbalaria showed 9.95 ± 1.05% and 3.06 ± 0.28% of early/late apoptosis and V. anagalloides 8.29 ± 1.09% and 1.95 ± 0.36% of early/late apoptosis compared with control (for EO was 7.45 ± 1.01% and 0.54 ± 0.25%, and for HY was 4.91 ± 1.97% and 0.70 ± 0.09% of early/late apoptosis, respectively) on the T24 cancer cell line. Future research will include other Croatian species of the genus Veronica to gain a more complete insight into the biological activity of the volatile products of this genus for potential discovery of drugs based on natural plant extracts.

Quantitative analysis of arbutin and hydroquinone in strawberry tree (Arbutus unedo L., Ericaceae) leaves by gas chromatography-mass spectrometry.

The phenolic glycoside arbutin and its metabolite with uroantiseptic activity hydroquinone occur naturally in the leaves of various medicinal plants and spices. In this study, an extraction procedure coupled with gas chromatography-mass spectrometry (GC-MS) was developed to determine arbutin and hydroquinone content in strawberry tree (Arbutus unedo L., Ericaceae) leaves. The method showed good linearity (R2>0.9987) in the tested concentration range (0.5-200 µg mL(-1)), as well as good precision (RSD<5%), analytical recovery (96.2-98.0%), and sensitivity (limit of detection=0.009 and 0.004 µg mL(-1) for arbutin and hydroquinone, respectively). The results obtained by the validated GC-MS method corresponded well to those obtained by high performance liquid chromatography (HPLC) method. The proposed method was then applied for determining arbutin and hydroquinone content in methanolic leaf extracts. The amount of arbutin in the leaves collected on the island of Kolocep (6.82 mg g(-1) dry weight) was found to be higher (tpaired=43.57, tc=2.92) in comparison to the amount of arbutin in the leaves collected on the island of Mali Losinj (2.75 mg g(-1) dry weight). Hydroquinone was not detected in any of the samples. The analytical features of the proposed GC-MS method demonstrated that arbutin and hydroquinone could be determined alternatively by gas chromatography. Due to its wide concentration range, the method could also be suitable for arbutin and hydroquinone analysis in leaves of other plant families (Rosaceae, Lamiaceae, etc.).

Toxicity and antioxidant capacity of Frangula alnus Mill. bark and its active component emodin.

In the present study toxicity of Frangula alnus Mill. bark, widely used as laxative, was investigated. Human peripheral blood lymphocytes (HPBLs) were treated with F. alnus bark extract or emodin (emodin is bark component with laxative property), and cytotoxicity, genotoxicity and parameters of oxidative stress were assessed. Also, polyphenol content of bark extract and antioxidant activity of the extract and emodin measured by DPPH, ABTS and FRAP methods were examined. The bark extract (500 µg/ml) produced cell death and DNA damage, while level of ROS changed at 250 µg/ml. Emodin induced cell death and DNA damage at 150 µg/ml and 200 µg/ml, respectively, and the increase of ROS was observed at 25 µg/ml. These results suggest that both, bark extract and emodin, are cyto/genotoxic to HPBLs and that oxidative stress is involved in the mechanism of their toxicity. The results on antioxidant activity showed that, unlike emodin, bark extract possess moderate antioxidant capacity (44.6%, 46.8% and 2.25 mmol Fe(2+)/g measured by DPPH, ABTS and FRAP assay, respectively) that can be related to relatively high phenolic content (116.07 mg/g). However, due to toxicological properties use of F. alnus bark as well as emodin-containing preparations should be taken with caution.

Antioxidant and Antimicrobial Properties of Veronica spicata L. (Plantaginaceae).

Tea made from Veronica spicata L. (syn. Pseudolysimachion spicatum (L.) Opiz, family Plantaginaceae) herb is used in traditional medicine as expectorant for cough and throat rinsing. To get insight into chemical compounds of V. spicata, the essential oil content (analysed by GC-FID and GC-MS), the content of phenolic compounds (analysed by HPLC), the content of macroelements and trace elements (analysed by ICP-AES), quantity of total phenols and total flavonoids (analysed by UV/Vis spectrophotometer), and antioxidant and antimicrobial properties were investigated. The main compounds of the essential oil were phytol (21.13%), heptacosane (10.22%) and pentacosane (8.91%). The most abundant investigated macroelement was K (8261 mg/kg) while Fe was the most represented element (32.49 mg/kg) among investigated micronutrients. Ten phenolic compounds (chrysin, rutin, quercitrin, quercitrin, and cichoric, ferulic, protocatehuic, rosmarinic, syringic and tannic acid) were identified and quantified. Additionally, V. spicata extract demonstrated notable radical-scavenging and chelating properties. The bacterial and fungal strains used in study were found to be susceptible toward methanolic and ethyl-acetate extracts with MIC values between 1.25 and 5.00 mg/mL using microdilution method. Aquose extracts were found to be antimicrobial inactive.

Genetic and phytochemical variability of six Teucrium arduini L. populations and their antioxidant/prooxidant behaviour examined by biochemical, macromolecule- and cell-based approaches.

Teucrium arduini L., an Ilyric-Balcanic endemic species, has been reported for decades as a valuable plant used in traditional medicine for treating digestive disorders. The present study evaluated genetic and phytochemical variability of six T. arduini populations in order to determine factors that influence an accumulation of polyphenolic compounds. Results strongly suggest that a phytochemical variation was caused by environmental rather than genetic factors. T. arduini leaf extract from the locality Ucka, which accumulated significantly more polyphenolic phytochemicals in comparison to others, showed antioxidant activity in DNA and lipid bioassays. Furthermore, the same extract exhibited prooxidant behaviour at protein level and induce formation of reactive oxygen species in human laryngeal carcinoma cells causing cytotoxic activity, in a dose dependent manner. All the results of the present study suggested that T. arduini extract could be responsible for antioxidative/prooxidative mechanisms and would help in determination of optimal conditions for their ethnopharmacological use.

Antimicrobial and Antioxidant Properties of Satureja Montana L. and S. Subspicata Vis. (Lamiaceae).

Satureja montanaL. and S. subspicata Vis. (Lamiaceae) are used for centuries in traditional medicine of Balcanic people in the healing of the lymphatic nodule and respiratory system inflammation. In this paper the amount of total phenols and flavonoids (analyzed by UV/Vis spectrophotometry), phenolic compounds profile (analyzed by HPLC), antimicrobial and antioxidant activities were studied in samples collected in seven per species populations of S. montanaand S. subspicatain Croatia. Eight phenolic compounds (rutin, quercetin, caffeic, p-coumaric, ellagic, protocatehuic, rosmarinic, and syringic acid) were identified and quantified using HPLC in methanolic and ethanolic extracts. Results showed that both species contained polyphenolics and other antioxidant compounds with chelating and radical-scavenging properties. The extracts prepared from both species showed broad spectrum of antimicrobial activity on in vitrotested microbial species (Staphylococcus aureus, Escherichia coli, Candida albicans, C. dubliniensis, C. krusei, C. glabrata, C. parapsilosis, and Microsporum gypseum).

Molecular and cellular approach in the study of antioxidant/pro-oxidant properties of Micromeria croatica (Pers.) Schott.

Phytochemical composition and antioxidant activity of three wild populations of endemic Illyric-Balcanic species Micromeria croatica (Pers.) Schott have been evaluated with respect to plant organ and growing location. Multivariate analysis (principal component analysis) was performed to visualise (dis-)similarity among samples and identify the correlations between phytochemical variables that explain the most variability. The tested leaf extract from BaCic kuk locality exhibited protective effects against reactive oxygen species-induced damage of DNA and inhibition of lipid peroxidation, while it caused oxidative degradation of protein in the bovine serum albumin assay at higher concentrations. This extract also exhibited cytotoxic activity and facilitated the formation of reactive oxygen species in the HEp2 cell line, in a dose-dependent manner.

Micromorphological traits and essential oil contents of Micromeria kerneri Murb. and M. juliana (L.) Benth. (Lamiaceae).

The chemical composition of the essential oil (analysed by GC and GC-MS), the types and distribution of trichomes and pollen morphology (analysed by scanning electron microscopy) were investigated in two closely related species, Micromeria kerneri Murb. and Micromeria juliana (L.) Benth. (Lamiaceae) from Southeast Europe as a contribution to their taxonomy. The essential oil of M. kerneri was characterized by a high concentration of oxygenated sesquiterpenes, with caryophyllene-oxide as the major compound. Caryophyllene-oxide was also the major component of the essential oil of M. juliana from all localities, except from Mt Krivosije (Montenegro), where piperitone oxide was the major constituent. Non-glandular trichomes, peltate trichomes, and two types of capitate trichomes (type 1 composed of one basal epidermal cell, and one head cell with subcuticular space; type 2 composed of one basal epidermal cell, two stalk cells, and one head cell with subcuticular space) were observed on leaves, the calyx and on the stem. Pollen of both species had six apertures (hexacolpate) set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation. Multivariate analysis (PCA and UPGMA) of essential oil components clearly separated the investigated M. kerneri and M. juliana populations, and confirmed the opinion that they are different taxa. On the other hand, micromorphological traits between these species were the same. Nevertheless, definitive conclusions about the taxonomic relationships among these species will require genetic analysis.

Hydroxytyrosol expresses antifungal activity in vitro.

Hydroxytyrosol (HT) is a potent antioxidant found in olive oil and leaves. Using several in vitro approaches, we tested antifungal activity of HT. HT showed broad spectrum of antifungal activity against medically important yeasts and dermatophyte strains with MIC values ranging between 97.6 µgml⁻¹ and 6.25 mgml⁻¹. The antimicrobial activity of HT was also tested using the time-kill methodology. Below the MIC value, HT showed potent damage of cell wall of Candida albicans ATCC 10231 using fluorescent dye-exclusion method. At the subinhibitory concentration, HT also influenced dimorphic transition of Candida indicating that HT is inhibitor of germ-tube formation as one of the most important virulence factor of C. albicans. Furthermore, HT showed disturbances in cell surface hydrophobicity (CSH) of C. albicans. The in vitro results indicate that HT caused a significant cell wall damage and changes in CSH as well as inhibition of germ-tube formation as virulence factor of C. albicans. The study indicates that HT has a considerable in vitro antifungal activity against medically important yeasts.

Antimicrobial activity of Willowherb (Epilobium angustifolium L.) leaves and flowers.

Since the aetiology of benign prostatic hyperplasia (BHP) is still unknown, the use of medicinal herb extracts and products prepared thereof are recommended due to their antimicrobial activity, especially during early stages of BHP. A comparison was performed of the in vitro antimicrobial activity (using broth microdilution assay) of flowers and leaves of willowherb (Epilobium angustifolium L., Onagraceae) from Mt. Velebit (Croatia). The strains (standard ATCC and clinical isolates) of Staphylococcus aureus (including MRSA), Bacillus subtilis, Escherichia coli (including p-fimbriae positive strain), Pseudomonas aeruginosa, Proteus mirabilis, Candida albicans, C. tropicalis, C. dubliniensis and Saccharomyces cerevisiae were susceptible with MIC values between 4.6±0.2 and 18.2±0.8 mg/mL. The results of in vitro studies showed that no differences were found in the antimicrobial activity between the ethanol extracts of leaves and flowers of E. angustifolium. Using the quantitative fluorescent assay with ethidium bromide and acridine orange, the viability of C. albicans ATCC 10231 was assessed after in vitro exposure to E. angustifolium leaf and flower ethanol extracts. Apoptosis of C. albicans blastospores dominated over necrosis in all treated samples after short-term exposure with 6 to 12 mg/mL of extracts. In addition to the valuable biological activity of E. angustifolium extracts, the data obtained from the in vitro diffusion, the dilution assay and antifungal viability fluorescent assay suggest that leaf and flower ethanol extracts of E. angustifolium L. are a promising complementary herbal therapy of conditions such as BHP.

Investigation of chemical compounds, antioxidant and antimicrobial properties of teucrium arduini L. (lamiaceae).

In this paper chemical composition of the essential oil (analysed by GC and GC-MS), the content of phenolic compounds (analysed by HPLC), quantity of total phenols and total flavonoids (analysed by UV/Vis spectrophotometer), antioxidant and antimicrobial activities of ethanolic extracts were investigated in endemic Teucrium arduini L. in population of Mt Biokovo (Croatia). The oil was characterized by a high concentration of sesquiterpene hydrocarbons (70.4%) of which ß-caryophyllene (35.2%) and germacrene D (18.7%) being the major compounds. Three phenolic compounds (quercetin, ferulic acid and rosmarinic acid) were identified and quantified in ethanolic extract of T. arduini using HPLC. The results also showed that T. arduini is a source of polyphenolic and other antioxidants with radical-scavenging and chelating properties. The ethanol extracts prepared from the leaf of T. arduini showed broad spectrum of antimicrobial activity on Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis, which are susceptible on concentration below or equal to 4.00 mg/mL, whilst Microsporum gypseum was resistant at investigated concentrations.

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, ß-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.

Chemical composition and antiphytoviral activity of essential oil of Micromeria graeca.

Phytochemical analysis of the essential oils of Micromeria graeca (L.) Rchb from Vis and Komiza on the island of Vis (Croatia) showed them to have a similar chemical composition. The oxygenated sesquiterpene alpha-bisabolol was a major component of both oils with percentages of 13.9% in the oil from Vis and 15.5% in the oil from Komiza. A comparison of the mean number of lesions on the essential oil-treated Chenopodium quinoa plants infected with satellite-associated cucumber mosaic virus (satCMV) with the corresponding control showed that the oil from Vis caused delay in the development and reduction of the number of lesions by 59.3%. The essential oil of M. graeca from Komiza was not effective in the reduction of satCMV infection.

Re-investigation of the anthraquinone pool of Rhamnus spp.: madagascin from the fruits of Rhamnus cathartica and R. intermedia.

As a continuation of our ongoing studies aimed at revealing the presence of oxyprenylated anthraquinones in plants belonging to the genus Rhamnus, in this paper we describe the extraction and HPLC separation of madagascin (3-isopentenyloxyemodin) from the fruits of R. cathartica L. and R. intermedia Steud. and Hochst. The title compound is described herein for the first time as a component of fruit extracts of Rhamnus species.

Micromorphological and chemotaxonomical traits of Micromeria croatica (Pers.) Schott.

A study on the types and distribution of trichomes, pollen morphology, chemical composition of essential oil (analyzed by GC-FID and GC/MS), and the content of macroelements (Na, K, Ca, and Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, and As) analyzed by ICP-AES (=inductively coupled plasma atomic emission spectroscopy) was conducted on Micromeria croatica (Pers.) Schott. Non-glandular trichomes, peltate trichomes (typical hairs of Lamiaceae), small capitate trichomes (composed of one basal epidermal cell and one head cell), and larger capitate trichomes (composed of one basal epidermal cell, two to three stalk cells, and one head cell with subcuticular space) were observed on leaves, stems, the calyx, and corolla. Pollen of M. croatica had six apertures (hexacolpate) which were set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation (hetrobrochate type). The essential oil contained from 27 to 39 constituents, the most representative of which were caryophyllene oxide and E-caryophyllene. Among the investigated macroelements, the content of K was highest (8730-10080 mg/kg). The content of trace elements ranged from 0.12 mg/kg (Cr) to 78.00 mg/kg (Fe), while the content of Cd, Ni, Hg, and As were lower than the limit of quantification.

Development and application of high-performance liquid chromatography for the study of two new oxyprenylated anthraquinones produced by Rhamnus species.

Rhamnus spp. is known to contain biologically active anthraquinone secondary metabolites but the presence of oxyprenylated ones is not reported. To this aim, a new simple, and accurate analytical method was developed to reveal chemical fingerprint of these analytes in plant extracts. Plant samples were analysed after extraction with n-hexane (first step) and methanol (second step) using a C(18) column with a mobile phase composed of 35% of water:65% of methanol (both with 1% formic acid, v/v) at 0.7 mL min⁻¹ flow rate in gradient elution mode. For quantitative analyses, selective detection was performed at 435 nm. The limit of quantification (LOQ) was 0.5 µM, with the only exception of 3-geranyloxyemodin for which the LOQ value was 5.0 µM, and external matrix-matched standard curves showed linearity up to 125 µM. The within- and between-batch precision (RSD%) values ranged from 0.2% to 12.9% while within- and between-batch trueness (bias%) values ranged from 12.2% to 12.7%. The method was applied to evaluate for the first time the presence and the quantities of oxyprenylated anthraquinones in Rhamnus spp. barks as well as the anthraquinone profile of Rhamnus pumila Turra. The proposed method could be directly applied to the selective quantification of these analytes in natural sources.

Phytochemical and micromorphological traits of endemic Micromeria pseudocroatica (Lamiaceae).

Micromeria pseudocroatica Silic is an endemic species distributed in southern Croatia. Chemical composition of the essential oil (analysed by GC and GC-MS), and the content of macroelements and trace elements analysed by ICP-AES was conducted. Additionally, a study on the types and distribution of trichomes was done by scanning electron microscopy. The essential oil was characterized by a high concentration of oxygenated monoterpenes, whose major compounds are borneol (22.7% and 24.8%) and camphor (16.1% and 13.9%). The content of Ca was highest (13202.69 mg/kg and 13223.83 mg/kg) among the investigated macroelements, while Fe was the most represented element (100.68 and 326.48 mg/kg) among the micronutrients. The content of potentially toxic elements that is Se, Cu, and Hg was below the limit of quantification. Non-glandular trichomes, peltate trichomes, and two types of capitate trichomes (type 1: one basal epidermal cell, one head cell with subcuticular space; type 2: one basal epidermal cell, two stalk cells, and one head cell with subcuticular space) were observed on leaves, bracteoles, calyx, corolla and stem.

Anthraquinone profile, antioxidant and antimicrobial properties of bark extracts of Rhamnus catharticus and R. orbiculatus.

The anthraquinone profile, antioxidant and antimicrobial activities as well as the total phenol and total flavonoid contents were determined in methanol extracts of the barks of Rhamnus catharticus L. and R. orbiculatus Bornm. The most abundant anthraquinone derivatives in R. catharticus were physcion (67.8%) and emodin (26.2%), while R. orbiculatus contained mostly physcion (81.3%) and chrysophanol (14.6%). R. catharticus displayed better activity in the beta-carotene-linoleic acid assay, as well as chelating activity, whereas its activity in the reducing power assay was significantly lower than that of R. orbiculatus. Both methanol extracts showed antimicrobial activity against all microbial species tested (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, Aspergillus niger, Microsporum gypseum) with MIC values either equal to or lower than 2.50 mg/mL. R. catharticus and R. orbiculatus contained several anthranoid aglycones and their bark extracts demonstrated notable antioxidant and antimicrobial properties. The results obtained indicate the medicinal potential of these two species.