Analysis of Lemon Verbena Polyphenol Metabolome and Its Correlation with Oxidative Stress under Glucotoxic Conditions in Adipocyte.

Lemon verbena has been shown to ameliorate obesity-related oxidative stress, but the intracellular final effectors underlying its antioxidant activity are still unknown. The purpose of this study was to correlate the antioxidant capacity of plasma metabolites of lemon verbena (verbascoside, isoverbascoside, hydroxytyrosol, caffeic acid, ferulic acid, homoprotocatechuic acid, and luteolin-7-diglucuronide) with their uptake and intracellular metabolism in hypertrophic adipocytes under glucotoxic conditions. To this end, intracellular ROS levels were measured, and the intracellular metabolites were identified and quantified by high-performance liquid chromatography with a diode array detector coupled to mass spectrometry (HPLC-DAD-MS). The results showed that the plasma metabolites of lemon verbena are absorbed by adipocytes and metabolized through phase II reactions and that the intracellular appearance of these metabolites correlates with the decrease in the level of glucotoxicity-induced oxidative stress. It is postulated that the biotransformation and accumulation of these metabolites in adipocytes contribute to the long-term antioxidant activity of the extract.

The influence of light quality on the production of bioactive metabolites - verbascoside, isoverbascoside and phenolic acids and the content of photosynthetic pigments in biomass of Verbena officinalis L. cultured in vitro.

In vitro callus cultures of Verbena officinalis L. were maintained on solid Murashige and Skoog medium, enriched with 1 mg dm-3 BA and 1 mg dm-3 IBA under LED lights (red, blue, red/blue 70%/30%), in darkness and under control fluorescent lamps. The measurements of 2 phenylpropanoid glycosides (verbascoside and isoverbascoside) and 23 phenolic acids were performed in methanolic extracts from the biomass collected after 2-, 3- and 4-week growth cycles using the HPLC-DAD method. The presence of verbascoside, isoverbascoside and additionaly 7 phenolic acids (protocatechuic, chlorogenic, vanillic, caffeic, ferulic, o-coumaric and m-coumaric acids) was confirmed in all extracts. Blue and red/blue lights stimulated the accumulation of verbascoside (max. of 6716 and 6023 mg 100 g-1 DW after a 4-week growth cycle) and isoverbascoside (max. 333 and 379 mg 100 g-1 DW also after 4 weeks). The maximum amounts of verbascoside and isoverbascoside were respectively 1.8- and 7.0-fold higher than under the control conditions. Phenolic acids were accumulated in different amounts, and the maximum total amounts ranged from 36 to 65 mg 100 g-1 DW. LED lights also stimulated their accumulation in comparison with darkness and control. The main phenolic acids included: m-coumaric acid (max. 39 mg 100 g-1 DW), ferulic acid (max. 12 mg 100 g-1 DW), and protocatechuic acid (max. 13 mg 100 g-1 DW). Additionally, the quantities of photosynthetic pigments (chlorophyll a, b and carotenoids) were estimated in acetonic extracts using spectrophotometry. Red/blue light stimulated the biosynthesis of pigments (max. total content 287 µg g-1 FW after 4-week growth cycles). This is the first study describing the effect of LED lights on the production of phenylpropanoid glycosides and phenolic acids in V. officinalis callus cultures. Very high amounts of verbascoside and isoverbascoside are interesting from a practical point of view.

Major, minor and trace element concentrations in spices and aromatic herbs from Sicily (Italy) and Mahdia (Tunisia) by ICP-MS and multivariate analysis.

Element contents in different types of spices and aromatic herbs collected from Italy and Tunisia were evaluated. The aim was to evaluate: the possible differences and/or similarities among the analyzed samples; if it is possible related the samples to their geographical origins; the nutritional quality and the potential health risks. Potassium, Ca, Mg and Ni were low in laurel and rosemary; mint and thyme showed the highest Na and the lowest Se contents; arsenic and Cd levels were found highest in verbena which had also the lowest Hg content; lastly, black pepper had the highest Mn and the lowest Pb contents. Instead discrimination between Italian and Tunisian samples for each spices and aromatic herbs under analysis was achieved by PCA. Essential elements intake through samples was small. Levels of Pb in some samples exceed the maximum allowable level, but any safety risk for consumers is excluded.

Towards eco-friendly secondary plant metabolite quantitation: Ultra high performance supercritical fluid chromatography applied to common vervain (Verbena officinalis L.).

This report presents the first ultra high performance supercritical fluid chromatography diode array detector based assay for simultaneous determination of iridoid glucosides, flavonoid glucuronides, and phenylpropanoid glycosides in Verbena officinalis (Verbenaceae) extracts. Separation of the key metabolites was achieved in less than 7 min on an Acquity UPC2 Torus Diol column using a mobile phase gradient comprising subcritical carbon dioxide and methanol with 0.15% phosphoric acid. Method validation for seven selected marker compounds (hastatoside, verbenalin, apigenin-7-O-glucuronide, luteolin-7-O-glucuronide, apigenin-7-O-diglucuronide, verbascoside, and luteolin-7-O-diglucuronide) confirmed the assay to be sensitive, linear, precise, and accurate. Head-to-head comparison to an ultra high performance liquid chromatography comparator assay did prove the high orthogonality of the methods. Quantitative result equivalence was evaluated by Passing-Bablok-correlation and Bland-Altman-plot analysis. This cross-validation revealed, that one of the investigated marker compound peaks was contaminated in the ultra high performance liquid chromatography assay by a structurally related congener. Taken together, it was proven that the ultra high performance supercritical fluid chromatography instrument setup with its orthogonal selectivity is a true alternative to conventional reversed phase liquid chromatography in quantitative secondary metabolite analysis. For regulatory purposes, assay cross-validation with highly orthogonal methods seems a viable approach to avoid analyte overestimation due to coeluting, analytically indistinguishable contaminants.

Transcriptomic analysis of Verbena bonariensis roots in response to cadmium stress.

BACKGROUND: Cadmium (Cd) is a serious heavy metal (HM) soil pollutant. To alleviate or even eliminate HM pollution in soil, environmental-friendly methods are applied. One is that special plants are cultivated to absorb the HM in the contaminated soil. As an excellent economical plant with ornamental value and sound adaptability, V. bonariensis could be adapted to this very situation. In our study, the Cd tolerance in V. bonariensis was analyzed as well as an overall analysis of transcriptome. RESULTS: In this study, the tolerance of V. bonariensis to Cd stress was investigated in four aspects: germination, development, physiological changes, and molecular alterations. The results showed that as a non-hyperaccumulator, V. bonariensis did possess the Cd tolerance and the capability to concentration Cd. Under Cd stress, all 237, 866 transcripts and 191, 370 unigenes were constructed in the transcriptome data of V. bonariensis roots. The enrichment analysis of gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed that differentially expressed genes (DEGs) under Cd stress were predominately related to cell structure, reactive oxygen species (ROS) scavenging system, chelating reaction and secondary metabolites, transpiration and photosynthesis. DEGs encoding lignin synthesis, chalcone synthase (CHS) and anthocyanidin synthase (ANS) were prominent in V. bonariensis under Cd stress. The expression patterns of 10 DEGs, validated by quantitative real-time polymerase chain reaction (qRT-PCR), were in highly accordance with the RNA-Sequence (RNA-Seq) results. The novel strategies brought by our study was not only benefit for further studies on the tolerance of Cd and functional genomics in V. bonariensis, but also for the improvement molecular breeding and phytoremediation.

Application of benchtop and portable near-infrared spectrometers for predicting the optimum harvest time of Verbena officinalis.

This study examined the applicability of near-infrared (NIR) spectroscopy coupled with multivariate data analysis (MVA) to determine the ideal harvest time of Verbena officinalis. NIR analyses were performed non-invasively on the fresh plant material based on the quantification of the key constituents verbenalin and verbascoside. Vibrational spectroscopic measurements were performed applying a conventional NIR benchtop device as well as a laboratory independent handheld NIR spectrometer. A novel high performance liquid chromatography (HPLC) method was applied as a reference method. For both instruments partial least squares (PLS) regression models were established performing cross validations (CV) and test-set validations (TSV). Quality parameters obtained for the benchtop device revealed that the newly established NIR method enabled reliable quantifications of the main compounds verbenalin and verbascoside related to the dried and fresh plant material. The results of the miniaturised spectrometer revealed that accurate quantitative calibration models could be developed for verbascoside achieving a comparable prediction power to the benchtop device. PLS models for verbenalin were less precise suggesting the application of portable devices including a different spectral range and resolution. The work demonstrated the feasibility of NIR vibrational spectroscopy performing direct measurements on pharmaceutically relevant fresh plant material enabling a quick and simple determination of the ideal harvest time of Verbena officinalis.

Bioavailability of lemon verbena (Aloysia triphylla) polyphenols in rats: impact of colonic inflammation.

Lemon verbena (Aloysia triphylla) infusion, a widely consumed herbal tea, contains significant amounts of polyphenols such as flavone diglucuronides and phenylpropanoid glycosides (mainly verbascoside). We have recently shown that lemon verbena infusion offers beneficial effects against dextran sodium sulphate (DSS)-induced colonic inflammation in rats. The present study aimed to evaluate the bioavailability and intestinal absorption of polyphenols derived from lemon verbena infusion in both healthy and colitic rats. For this purpose, lemon verbena infusion was given to rats ad libitum for 14 d, and then 4 % DSS was added to the infusion for 7 d. Before and after DSS administration, 24 h urinary excretion of polyphenols was determined. Flavones were excreted in the urine as conjugated aglycones, and their excretion was not significantly altered by colonic inflammation. Only trace amounts of verbascoside were excreted in the urine, but various metabolites (hydroxycinnamic acids) were detected. The urinary excretion of hydroxycinnamic acids, particularly that of caffeic acid, increased after DSS administration (P< 0·05). Only flavone aglycones (luteolin and diosmetin) were excreted in the faeces in small proportions (3·2 % of ingested flavones). Intestinal absorption of lemon verbena polyphenols was examined using an in situ intestinal perfusion model. Intestinal absorption of verbascoside and flavone diglucuronides did not significantly differ between the healthy and colitic rats. Collectively, these results show that intestinal absorption and urinary excretion of lemon verbena flavone diglucuronides were not altered by colonic inflammation, but that urinary excretion of hydroxycinnamic acids derived from verbascoside was affected in a colitic situation.

Enhancement of phosphate absorption by garden plants by genetic engineering: a new tool for phytoremediation.

Although phosphorus is an essential factor for proper plant growth in natural environments, an excess of phosphate in water sources causes serious pollution. In this paper we describe transgenic plants which hyperaccumulate inorganic phosphate (Pi) and which may be used to reduce environmental water pollution by phytoremediation. AtPHR1, a transcription factor for a key regulator of the Pi starvation response in Arabidopsis thaliana, was overexpressed in the ornamental garden plants Torenia, Petunia, and Verbena. The transgenic plants showed hyperaccumulation of Pi in leaves and accelerated Pi absorption rates from hydroponic solutions. Large-scale hydroponic experiments indicated that the enhanced ability to absorb Pi in transgenic torenia (AtPHR1) was comparable to water hyacinth a plant that though is used for phytoremediation causes overgrowth problems.

Phytoremediation of a sulphonated azo dye Green HE4B by Glandularia pulchella (Sweet) Tronc. (Moss Verbena).

PURPOSE: The dyes and dye stuffs present in effluents released from textile dyeing industries are potentially mutagenic and carcinogenic. Phytoremediation technology can be used for remediating sites contaminated with such textile dyeing effluents. The purpose of the work was to explore the potential of Glandularia pulchella (Sweet) Tronc. to decolorize different textile dyes, textile dyeing effluent, and synthetic mixture of dyes. METHODS: Enzymatic analysis of the plant roots was performed before and after decolorization of dye Green HE4B. Analysis of the metabolites of Green HE4B degradation was done using UV-Vis spectroscopy, high-performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and gas chromatography-mass spectroscopy (GC-MS). The ability of the plant to decolorize and detoxify a textile dyeing effluent and a synthetic mixture of dyes was studied by a determination of the American Dye Manufacturer's Institute (ADMI), biological oxygen demand (BOD), and chemical oxygen demand (COD). Phytotoxicity studies were performed. RESULT: Induction of the activities of lignin peroxidase, laccase, tyrosinase, and 2,6-dichlorophenol indophenol reductase was obtained, suggesting their involvement in the dye degradation. UV-Vis spectroscopy, HPLC, and FTIR analysis confirmed the degradation of the dye. Three metabolites of the dye degradation were identified, namely, 1-(4-methylphenyl)-2-{7-[(Z)-phenyldiazenyl] naphthalen-2-yl} diazene; 7,8-diamino-2-(phenyldiazenyl) naphthalen-1-ol; and (Z)-1,1'-naphthalene-2,7-diylbis (phenyldiazene) using GC-MS. ADMI, BOD, and COD values were reduced. The non-toxic nature of the metabolites of Green HE4B degradation was revealed by phytotoxicity studies. CONCLUSION: This study explored the phytoremediation ability of G. pulchella (Sweet) Tronc. in degrading Green HE4B into non-toxic metabolites.

The efficacy of ethanolic extract of Lemon verbena on the skin infection due to Staphylococcus aureus in an animal model.

Daily increasing of Staphylococcus aureus resistance to various antibiotics in particular penicillin and Methecilin has led the scientist to look fore new medicines in this area. In an in vitro laboratory studies, it has been demonstrated that ethanolic extract of Lemon verbena can prevent the growth of Staphylococcus aureus. In this study the efficacy of ethanolic extract of Lemon verbena against Staphylococcus aureus skin infection were assessed in an in vivo, in animal model. 200lambda of Staphylococcus aureus suspension, were inoculated intradermally on the shoulder of 63 laboratory 20-30 g mice. the mice were divided in to 4 groups, 2 control groups: Negative (without treatment) and positive (treated with Mupirucin) and 2 test groups that treated for 7 days by ointment prepared from ethanolic extract of Lemon verbena (group 3), or injection of Lemon verbena solution (group 4). The status of wounds, the rate of recovery was studied and the presence of local pus after dissection of mice on day 8 recorded and compared with each other. The wound appearance in the second day, on the injection site of S. aureus, in Group 1, 4, 3 and 2 were 84.2, 66.7, 46.2 and 23.1%, respectively. In the final day, the lesion still was remained in 78.9, 23.1, 92.3 and 77.7% in groups 1 -4, respectively. The necrotic and wide wounds were more observed in groups 1 and 3 vs two other groups. The results from this investigation indicate that the ointment prepared from ethanolic extract of Lemon verbena is a proper medication to prevent the skin infection by Staphylococcus aureus in early phase.