Measurement of Histamine in Stinging Insect Venoms by Isotachophoresis

Abstract

An isotachophoretic histamine assay has been developed to determine the histamine content in hymenoptera stinging insect venoms. The amount of histamine was lowest in bee venom (0.9%) while the histamine content of vespid venom extracts varied between 2.7–5.2%. Histamine, determined quantitatively by isotachophoresis, correlated well with the histamine values achieved with reversed-phase high performance liquid chromatography.     

Urtica dioica Agglutinin (UDA) – Separation and Quantification of Individual Isolectins by Reversed Phase High Performance Liquid Chromatography

Summary Stinging nettle (Urtica dioica and Urtica urens) roots are traditionally used as a remedy against BPH in Europe, and one of the plant's active principle discussed is UDA (a mixture of different isolectins). This paper reports on the first HPLC-method permitting the qualitative and quantitative analysis of individual isolectins in Urtica plant material. Optimum results were obtained by using a cyano column, and a mobile phase comprising of 10 mM phosphate buffer (pH 6.75) and acetonitrile. Temperature and detection wavelength were adjusted to 25 °C and 228 nm, respectively. The method was successfully validated for linearity, accuracy and precision. Several U. dioica samples were analysed, and except for one, all contained lectins (isolectins II, I, V and VI in varying composition). Individual compounds were assigned by comparison with reference material or based on data obtained from LC-MS experiments. The quantitative results showed variations in the lectin content from 0.016 to 0.401%; in three commercial products, which were additionally analyzed, no lectins were found.     

Isotachophoretic Analysis of Yellow Jacket Vespula Species Venom. Identification of Separated Components by Zymography,Electrophoresis and Immunological Analysis

Abstract

Isotachophoresis, with its inherent high resolving power is a rapid and powerful technique for characterization of Hymenoptera insect venoms. By applying a combination of isotachophoresis for the first dimension separation step, and enzyme based assays, electrophoresis or immunological assays for the second dimension identification, it has been possible to identify the most important peptide/protein components in “Yellow Jacket” (Vespula species) venom. The combined two dimensional procedure is rapid, reproducible and well suited for determination of the antigenic/allergenic composition of various Hymenoptera venoms.     

Phenolic Constituents of Lamium album L. subsp. album Flowers: Anatomical,Histochemical, and Phytochemical Study

Flos Lamii albi has a high biological activity and is widely used in herbal medicine. The aim of the study was to characterize the secretory structures present in Lamium album subsp. album corolla and the location of phenolic compounds. Additionally, we carried out qualitative phytochemical analyses of flavonoids and phenolic acids. Light, fluorescence, and scanning electron microscopy were used to analyze the structure of the floral organs. The main classes of phenolic compounds and their localization were determined histochemically. Phytochemical analyses were performed with high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). Six types of glandular trichomes were found which contained flavonoids, phenolic acids, and tannins. The phytochemical studies demonstrated the presence of caffeic, chlorogenic, ferulic, gallic, p-coumaric, protocatechuic, syringic, gentisic, and vanillic phenolic acids as well as rutoside, isoquercetin, and quercetin flavonoids. The corolla in L. album subsp. album has antioxidant properties due to the presence of various polyphenols, as shown by the histo- and phytochemical analyses. The distribution and morphology of trichomes and the content of phenolic compounds in the corolla have taxonomic, pharmacognostic, and practical importance, facilitating the identification of the raw material.     

Variation in the Content of Bioactive Compounds in Infusions Prepared from Different Parts of Wild Polish Stinging Nettle (Urtica dioica L.)

Nettle is a common plant that offers many health benefits and is grown all over the world. The content of active compounds in roots, stems, and leaves was determined based on the extraction procedure optimized using the Central Composite Design. Flavonols, phenolic acids, trigonelline, nicotinamide, nicotinic acids, and short-chain organic acids were determined with the use of LC–MS/MS and capillary isotachophoresis. Trigonelline, which was not previously reported in the roots and stems of nettle, was found in all parts of the plant and considerable variations in its content were observed (2.8–108 µg g⁻¹). Furthermore, the Principal Component Analysis taking into account more variables demonstrated differences in the content of bioactive components between roots and aerial parts of nettle.     

Phenolic Profile,Antioxidant Capacity and Antimicrobial Activity of Nettle Leaves Extracts Obtained by Advanced Extraction Techniques

Nettle is a widely known plant whose high biological activity and beneficial medicinal effects are attributed to various bioactive compounds, among which polyphenols play an important role. In order to isolate polyphenols and preserve their properties, advanced extraction techniques have been applied to overcome the drawbacks of conventional ones. Therefore, microwave-assisted extraction (MAE) has been optimized for the isolation of nettle leaves polyphenols and it was compared to pressurized liquid extraction (PLE) and conventional heat-reflux extraction (CE). The obtained extracts were analyzed for their individual phenolic profile by UPLC MS² and for their antioxidant capacity by ORAC assay. MAE proved to be the more specific technique for the isolation of individual phenolic compounds, while PLE produced extracts with higher amount of total phenols and higher antioxidant capacity. Both techniques were more effective compared to CE. PLE nettle extract showed antimicrobial activity against bacteria, especially against Gram-negative Pseudomonas fragi ATCC 4973 and Campylobacter jejuni NCTC 11168 strains. This suggests that PLE is suitable for obtaining a nettle extract with antioxidant and antimicrobial potential, which as such has great potential for use as a value-added ingredient in the food and pharmaceutical industry.     

Urtica dioica agglutinin: separation, identification, and quantitation of individual isolectins by capillary electrophoresis and capillary electrophoresis-mass spectrometry

With benign prostatic hyperplasia (BPH) being a major health problem in ageing men, alternative therapeutic approaches (e.g., with phytopharmaceuticals) are of great interest. Based on pharmacological evidences, one of the most promising options in that respect are the lectins found in Urtica dioica (stinging nettle) roots. In this study the qualitative and quantitative analysis of individual isolectins in U. dioica extracts is described, which is the first report on using capillary electrophoresis (CE) for the analysis of lectins in plant material at all. By utilizing a 200 mM sodium acetate buffer (pH 3.75) a baseline separation and determination of four closely related isolectins was feasible within 20 min in the aqueous plant extracts. The individual compounds were identified based on reference compounds as well as data obtained from CE-mass spectrometry (MS) experiments. After modifying the optimized CE conditions to 100 mM ammonium formate buffer with pH 3.75 and a voltage of 15 kV, the isolectins were clearly assignable in positive electrospray ionization (ESI) mode. The quantitative results obtained by CE (the total lectin content varied from 0 to 0.42% in the samples) were accurate (recovery rates of spiked samples between 92.5 and 96.2%), precise (relative standard deviation < 5%) and in good agreement to those obtained by High-performance liquid chromatography (HPLC). As for peak resolution, assignable compounds and required separation time the newly developed CE method was clearly advantageous over the determination achieved by LC.     

Determining the Parameters of the Stinging Nettle (Urtica dioica L.) Hydrolate Distillation Process

Stinging nettle (Urtica dioica L.) is a common perennial herb well known for its therapeutic, cosmetic and food use. Despite the popularity of nettle hydrolate, there is currently no literature describing its composition; likewise, there is still a lack of research describing in detail the parameters of hydrolates in general. U. dioica hydrolate fractions were obtained by industrial steam distillation of fresh herb. Total stinging nettle hydrolate was prepared by mixing an equal volume of each fraction. The volatiles were isolated from hydrolate samples by liquid–liquid extraction with diethyl ether, and analysed using GC-FID-MS. Over eighty volatile compounds were identified in U. dioica hydrolate. The main group of constituents were oxygenated compounds, mainly alcohols (e.g., (E)- and (Z)-hex-3-en-1-ol, carvacrol) and oxides (e.g., caryophyllene oxide). The content of volatiles in the representative sample of total hydrolate amounted to 58.2 mg/L. Some qualitative and quantitative changes in the composition of U. dioica hydrolate were observed during the progress of distillation. The content of low chain aliphatic alcohols ((E)- and (Z)-hex-3-en-1-ol) decreased, whereas the percentage of some monoterpene alcohols (carvacrol and α-terpineol) increased. The total content of volatiles in hydrolate also changed and decreased (128.0–6.2 mg/L) during distillation progress. According to our results, to produce stinging nettle hydrolate of good quality, the proper relationship between the amount of hydrolate and raw plant material should result in obtaining 0.74 L hydrolate from 1 kg of fresh stinging nettle herb. Therefore, it may be assumed that the high alcohol content may increase the microbiological stability of the product.