Methodological approach to determine carlina oxide - a main volatile constituent of Carlina acaulis L. essential oil.

In this work, a fast and low-cost voltammetric methodology for determination of carlina oxide in plant extracts was developed. The best results were obtained using a boron-doped diamond electrode (BDDE). The voltammetric measurements of carlina oxide were performed in a 0.1 mol/L solution of sulphuric acid. After 30 s of stirring the solution, differential pulse voltammograms (DPVs) were recorded from 0.5 to 1.8 V. The amplitude was 75 mV and the scan rate was 175 mV/s. Measurements were recorded in non-deaerated solutions. The background current was subtracted from each registered voltammogram; then they were cut from 0.5 to 1.5 V. The detection and quantification limits were 0.28 and 0.93 µg/L, respectively, and repeatability expressed as the relative standard deviation of 0.1 mg/L of carlina oxide was 1.9% (n = 5). The results were compared with those obtained using gas chromatography with a flame ionization detector and high performance liquid chromatography with a photodiode array detector.

Systematic Evaluation of Chromatographic Parameters for Isoquinoline Alkaloids on XB-C18 Core-Shell Column Using Different Mobile Phase Compositions.

Chelidonium majus L. is a rich source of isoquinoline alkaloids with confirmed anti-inflammatory, choleretic, spasmolytic, antitumor, and antimicrobial activities. However, their chromatographic analysis is difficult because they may exist both in charged and uncharged forms and may result in the irregular peak shape and the decrease in chromatographic system efficacy. In the present work, the separation of main C. majus alkaloids was optimized using a new-generation XB-C18 endcapped core-shell column dedicated for analysis of alkaline compounds. The influence of organic modifier concentration, addition of salts, and pH of eluents on chromatographic parameters such as retention, resolution, chromatographic plate numbers, and peak asymmetry was investigated. The results were applied to elaborate the optimal chromatographic system for simultaneous quantification of seven alkaloids from the root, herb, and fruit of C. majus.

Application of Raman spectroscopy for direct analysis of Carlina acanthifolia subsp. utzka root essential oil.

Carlina genus plants e.g. Carlina acanthifolia subsp. utzka have been still used in folk medicine of many European countries and its biological activity is mostly associated with root essential oils. In the present paper, Raman spectroscopy (RS) was applied for the first time for evaluation of essential oil distribution in root of C. acnthifolia subsp. utzka and identification of root structures containing the essential oil. Furthermore, RS technique was applied to assess chemical stability of oil during drying of plant material or distillation process. Gas chromatography-mass spectrometry was used for qualitative and quantitative analysis of the essential oil. The identity of compounds was confirmed using Raman, ATR-IR and NMR spectroscopy. Carlina oxide was found to be the main component of the oil (98.96% ± 0.15). The spectroscopic study showed the high stability of essential oil and Raman distribution analysis indicated that the oil reservoirs were localized mostly in the structures of outer layer of the root while the inner part showed nearly no signal assigned to the oil. Raman spectroscopy technique enabled rapid, non-destructive direct analysis of plant material with minimal sample preparation and allowed straightforward, unambiguous identification of the essential oil in the sample.

In Vitro Antiproliferative Activity of Extracts of Carlina acaulis subsp. caulescens and Carlina acanthifolia subsp. utzka.

Various species of the Carlina genus have been used in traditional medicine in many countries to treat numerous skin disorders, including cancer. The objective of this work was to assess the anticancer properties of root and leaf extracts from Carlina acaulis subsp. caulescens and C. acanthifolia subsp. utzka. Anti-tumor properties of the extracts were explored using a tetrazolium-based cell viability assay and flow cytometric apoptosis analysis, followed by immunodetection of phosphoactive ERK1/2 in UACC-903, C32, and UACC-647 human melanoma cell lines. Normal human fibroblasts were used as a control. Leaf extracts inhibited the viability of all tested melanoma cell lines in a dose-dependent fashion while the fibroblasts were less sensitive to such extract. The root extracts inhibited the proliferation of UACC-903 and UACC-647 cells only at the highest doses (300 µg/mL). However, the C32 and fibroblast cells exhibited an increase in the cellular proliferation rate and no caspase activity was observed in response to the root extracts (100 µg/mL). An increase in caspase activity was observed in melanoma cells treated with the leaf extracts of both Carlina species. Leaf extracts from C. acaulis subsp. caulescens (100 µg/mL) inhibited proliferatory ERK1/2 in UACC-903 and C32 cells, as demonstrated by the decrease in ERK1/2 phosphorylation. No reduction in phospho-ERK1/2 was observed in the tested cell lines treated with the root extracts, apart from UACC-647 after incubation with the C. acanthifolia subsp. utzka root extract (100 µg/mL). There was no change in ERK1/2 phosphorylation in the fibroblasts. The extracts from the leaves and roots were analyzed by HPLC and the analysis showed the presence of triterpenes and phenolic acids as the main extract components. The research demonstrated that the extracts from the leaves of the plants were cytotoxic against the human melanoma line and induced apoptosis of the cells. The triterpene fraction present in the tested extracts may be responsible for this activity.

Evaluation of seasonal changes of triterpenic acid contents in Viscum album from different host trees.

CONTEXT: Viscum album L. (Loranthaceae) is a semi-parasitic plant used in pharmacy and medicine mostly for its hypotensive and anticancer activity. The effects may be related to the presence of triterpenic acids, such as betulinic (BA) and oleanolic (OA) acids. OBJECTIVES: In our investigations the content of triterpenic acids in V. album from different host trees depending on the season of harvest was determined. MATERIAL AND METHODS: V. album herb was dried and extracted with ethyl acetate using ultrasound energy. The reversed phase HPLC-PDA method was used for the analysis of triterpenic acids. The structure of the target components was confirmed by mass spectrometry with an electrospray ionization source. RESULTS: Diversity in the content of both compounds was noted; however, OA was the dominant triterpenic acid and the amount thereof was ∼10 times higher than that of BA. The analysis of changes in the amount of triterpenic acids during the spring-winter period revealed the highest content of OA in summer (from 6.84 to 13.65 mg/g). In turn, in the other seasons of harvest, the content was in the range of 4.41-9.83, 6.41-9.56 and 5.59-12.16 mg/g for spring, autumn and winter, respectively. In most cases, a similar tendency was observed for BA. DISCUSSION AND CONCLUSION: In most cases, the highest amount of the investigated compounds was found in summer; thus, this period seems to be optimal for acquisition of plant material rich in triterpenic acids.

The Stimulatory Effect of Strontium Ions on Phytoestrogens Content in Glycine max (L.) Merr.

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

Herbal preparation extract for skin after radiotherapy treatment. Part One--Preclinical tests.

Naran R is a herbal composition made of Plantago lanceolate folium, Malvae arboreae flos, Calendulae flos, Chamomillae inflorescentia, Lamii albi flos to prepare compresses or to wash skin with inflammations. The extract of this preparation is mixed to be applied as an ointment on patients' skin after radiotherapy. Experiments performed in vitro are part of pre-clinical tests with Naran R ointment. This study examined the impact of the plant composition for ethanol-water extract on human skin fibroblasts (HSF) culture. Samples of extract, prepared from patented amounts of herbs, were in the range of 25-225 µg/mL. Six methods were applied: standard spectrophotometric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, neutral red (NR) uptake assay, DPPH free radical scavenging test, labeling of cytoskeleton F-actin, staining of argyrophilic nucleolar organizer regions (AgNORs) and trypan blue coloration. The extract concentration 75 µg/mL was established as safe for application on human skin. In labeling of F-actin with rhodamine-phalloidin dye at this concentration the cytoskeleton was stable. The extract did not influence the membrane stability and had positive influence on the proliferation activity. It was confirmed in AgNOR test during incubation with extract, which led to formation of larger amount of smaller nucleolins. In DPPH scavenging activity test, the extract revealed over 8% higher free-radical scavenging activity in comparison to control. After trypan blue staining, the extract in concentration 125 µg/mL significantly lowered the cell viability. When the cytotoxic and anti-proliferative activity of the extracts were analyzed, MTT and Neutral Red (NR) methods were used. The cells' viability was maintained on a constant level (80-110%) after 24, 48 and 72 h of incubation. During all time of NR test (72 h) and even when 225 µg/mL of extract was applied, the viability of cells was in range 80-110% of control. Positive influence of the extract on investigated cells structure and proliferation, lack of toxicity and increasing anti-oxidant activity enable to consider this preparation as a natural remedy with potential application in skin therapy after radiation.

HPTLC-densitometry determination of triterpenic acids in Origanum vulgare, Rosmarinus officinalis and Syzygium aromaticum.

Spices play an important role in the chemoprevention and they can be a rich source of biologically active compounds such as triterpenes in the human diet. A method based on high performance thin-layer chromatography combined with densitometry for determination of ursolic and oleanolic acids in some common spices was elaborated. The prechromatographic derivatization with 1% of iodine solution was used to enable simultaneous analysis of these triterpenes. The extracts were separated on HPTLC silica gel 60 F254 plates with use of mobile phase consisting of toluene-petroleum ether-ethyl acetate-acetonitrile 5: 5: 1: 0.3 (v/v/v/v). After drying, the plates were sprayed with 10% (v/v) ethanol solution of sulfuric acid (VI) and heated to 120 degrees C for 3 min. Quantification was performed in fluorescence/reflectance mode at a wavelength of 400 nm using a computer-controlled densitometer Desaga CD 60.