Microextraction of Reseda luteola-Dyed Wool and Qualitative Analysis of Its Flavones by UHPLC-UV, NMR and MS.

Detailed knowledge on natural dyes is important for agronomy and quality control as well as the fastness, stability, and analysis of dyed textiles. Weld (Reseda luteola L.), which is a source of flavone-based yellow dye, is the focus of this study. One aim was to reduce the required amount of dyed textile to ≤50 µg for a successful chromatographic analysis. The second aim was to unambiguously confirm the identity of all weld flavones. By carrying out the extraction of 50 µg dyed wool with 25 µL of solvent and analysis by reversed-phase UHPLC at 345 nm, reproducible chromatographic fingerprints could be obtained with good signal to noise ratios. Ten baseline separated peaks with relative areas ≥1% were separated in 6 min. Through repeated polyamide column chromatography and prepHPLC, the compounds corresponding with the fingerprint peaks were purified from dried weld. Each was unequivocally identified, including the position and configuration of attached sugars, by means of 1D and 2D NMR and high-resolution MS. Apigenin-4'-O-glucoside and luteolin-4'-O-glucoside were additionally identified as two trace flavones co-eluting with other flavone glucosides, the former for the first time in weld. The microextraction might be extended to other used dye plants, thus reducing the required amount of precious historical textiles.

In vitro antibacterial activities of compounds isolated from roots of Caylusea abyssinica.

BACKGROUND: Caylusea absyssinica, a plant used as vegetable and for medicinal purposes was selected for in vitro antibacterial evaluation in this study. The main aim of this study was to isolate compounds from the plant roots and evaluate their antibacterial activities on clinical bacterial test strains. METHODS: Compounds from roots of Caylusea absyssinica (fresen) were identified based on observed spectral (1H-NMR, 13C-NMR and IR) data and physical properties (melting point) as well as reported literature. Disk diffusion method was employed to evaluate the antibacterial activities of the isolated compounds on four test bacterial strains namely, Staphylococcus aureus (ATCC25903), Escherichia coli (ATCC25722), Pseudomonas aeruginosa (DSMZ1117) and Salmonella thyphimurium (ATCC13311). RESULTS: Two compounds, CA1 and CA2 were isolated from the methanol crude extract of the roots of Caylusea absyssinica (fresen). The compounds were identified as ß-sitosterol and stigmasterol, respectively. Evaluation of antibacterial activities revealed that the compounds are active against all the bacterial strains in the experiment, showing inhibition zones ranging from 12 mm-15 mm by CA1 and 11 mm-18 mm by CA2 against the different test strains. However, the compounds were less active than the reference drug (Gentamycine), which showed minimum inhibition zone of 21 mm (Pseudomonas aeruginosa) and maximum of 28 mm (Escherichia coli) inhibition zone. DISCUSSION AND CONCLUSION: The isolation of the compounds is the first report from roots of Caylusea abyssinica and could be potential candidates for future antibacterial drug development programs.

Cytotoxic effect of Reseda lutea L.: A case of forgotten remedy.

ETHNOPHARMACOLOGICAL RELEVANCE: Reseda lutea L. (Resedaceae) or Wild Mignonette is a widely distributed plant species. Pliny the Elder (AD 23-AD 79), a Roman scholar and naturalist, reported the use of R. lutea for reducing tumors in his Historia naturalis. Accounts of the beneficial effects of R. lutea in tumor treatment could also be found in the works of later authors, such as Étienne François Geoffroy (1672-1731) and Samuel Frederick Gray (1766-1828). However, to date no in vivo or in vitro evidence exists in support of the alleged tumor healing properties of R. lutea. MATERIALS AND METHODS: The composition of autolysates obtained from different organs (root, flower and fruit) of R. lutea was investigated by GC and GC-MS analyses and IR, 1D and 2D NMR spectroscopy. These analyses led to the discovery of a new compound isolated in pure form from the flower autolysate. Autolysates and their major constituents were submitted to MTT-dye reduction cytotoxic assay on human A375 (melanoma) and MRC5 (fibroblast) cell lines. Mechanism of the cytotoxic effects was studied by cell cycle analysis and Annexin V assay. RESULTS: Benzyl isothiocyanate and 2-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate were identified as the major constituents of the root and flower autolysates, respectively (the later represents a new natural product). These compounds showed significant antiproliferative effects against both cell lines, which could also explain the observed high cytotoxic activity of the tested autolysates. Cell cycle analysis revealed apoptosis as the probable mechanism of cell death. CONCLUSIONS: Tumor healing properties attributed to R. lutea in the pre-modern texts were substantiated by the herein obtained results. Two isothiocyanates were found to be the major carriers of the observed activity. Although there was a relatively low differential effect of the plant metabolites on transformed and non-transformed cell lines, one can argue that the noted strong cytotoxicity provides first evidence that could explain the long forgotten use of this particular species.

The photoprotective and antioxidative properties of luteolin are synergistically augmented by tocopherol and ubiquinone.

Ultraviolet radiation induces DNA damage and oxidative stress which can result in skin inflammation, photoaging, and photocarcinogenesis. The flavonoid luteolin that is present in high amounts in the dyers weld, Reseda luteola, is one of the most potent antioxidative plant metabolites and also has ultraviolet-absorbing properties.The aim of this study was to determine whether tocopherol and ubiquinone add synergistic antioxidative values to luteolin. None of the substances showed cytotoxic effects in concentrations from 0.25 to 4 µg/mL. The photoprotective and antioxidant effect of equivalent concentrations of luteolin, tocopherol, and ubiquinone and their combination in a ratio of 4 : 4 : 1 were studied in solar simulator irradiated human skin fibroblasts. Luteolin had a half-maximal radical scavenging concentration of 2 µg/mL, whereas tocopherol and ubiquinone were only effective at higher concentrations. None of the substances showed a phototoxic effect, and only luteolin had a moderate photoprotective effect at 2 µg/mL. The combination of luteolin, tocopherol, and ubiquinone exerted a synergistic radical scavenging effect already at a concentration of 0.25 µg/mL and a complete photoprotection at 2 µg/mL.In summary, our findings suggest that the potent antioxidant and photoprotective effect of flavonoids like luteolin may be further increased by the addition of low concentrations of other antioxidants such as tocopherol and ubiquinone.

Physiological and behavioural effects of fruit toxins on seed-predating versus seed-dispersing congeneric rodents.

Fleshy, ripe fruits attract seed dispersers but also seed predators. Although many fruit consumers (legitimate seed dispersers as well as seed predators) are clearly exposed to plant secondary compounds (PSCs), their impact on the consumers' physiology and foraging behaviour has been largely overlooked. Here, we document the divergent behavioural and physiological responses to fruit consumption of three congeneric rodent species in the Middle East, representing both seed dispersers and seed predators. The fruit pulp of the desert plant Ochradenus baccatus contains high concentrations of glucosinolates (GLSs). These GLSs are hydrolyzed into active toxic compounds upon contact with the myrosinase enzyme released from seeds crushed during fruit consumption. Acomys russatus and A. cahirinus share a desert habitat. Acomys russatus acts as an O. baccatus seed predator, and A. cahirinus circumvents the activation of the GLSs by orally expelling vital seeds. We found that between the three species examined, A. russatus was physiologically most tolerant to whole fruit consumption and even A. minous, which is evolutionarily naïve to O. baccatus, exhibits greater tolerance to whole fruit consumption than A. cahirinus. However, like A. cahirinus, A. minous may also behaviourally avoid the activation of the GLSs by making a hole in the pulp and consuming only the seeds. Our findings demonstrate that seed predators have a higher physiological tolerance than seed dispersers when consuming fruits containing toxic PSCs. The findings also demonstrate the extreme ecological/evolutionary lability of this plant-animal symbiosis to shift from predation to mutualism and vice versa.

Evaluation of the effects of 80% methanolic leaf extract of Caylusea abyssinica (fresen.) fisch. & Mey. on glucose handling in normal, glucose loaded and diabetic rodents.

BACKGROUND: The leaves of Caylusea abyssinica (fresen.) Fisch. & Mey. (Resedaceae), a plant widely distributed in East African countries, have been used for management of diabetes mellitus in Ethiopian folklore medicine. However, its use has not been scientifically validated. The present study was undertaken to investigate antidiabetic effects of the hydroalcoholic leaf extract of C. abyssinica extract in rodents. MATERIALS AND METHOD: Male Animals were randomly divided into five groups for each diabetic, normoglycemic and oral glucose tolerance test (OGTT) studies. Group 1 served as controls and administered 2% Tween-80 in distilled water, (TW80); Group 2 received 5 mg/kg glibenclamide (GL5); Groups 3, 4 and 5 were given 100 (CA100), 200 (CA200) and 300 (CA300) mg/kg, respectively, of the hydroalcoholic extract of C. abyssinica. Blood samples were then collected at different time points to determine blood glucose levels (BGL). Data were analyzed using one way ANOVA followed by Dunnet's post hoc test and p < 0.05was considered as statistically significant. RESULTS: In normal mice, CA200 and GL5 induced hypoglycemia starting from the 2nd h but the hypoglycemic effect of CA300 was delayed and appeared at the 4th h (p < 0.05 in all cases). In diabetic mice, BGL was significantly reduced by CA100 (p < 0.05) and CA300 (p < 0.01) starting from the 3rd h, whereas CA200 (p < 0.001) and GL5 (p < 0.05) attained this effect as early as the 2nd h. In OGTT, TW80 (p < 0.01) and CA100 (p < 0.01) brought down BGL significantly at 120 min, while CA200 (p < 0.001) and GL5 (p < 0.001) achieved this effect at 60 min indicating the oral glucose load improving activity of the extract. By contrast, CA300 was observed to have no effect on OGTT. Acute toxicity study revealed the safety of the extract even at a dose of 2000 mg/kg. Preliminary phytochemical study demonstrated the presence of various secondary metabolites, including, among others, saponins, flavonoids and alkaloids. CONCLUSION: The results indicate that C. abyssinica is endowed with antidiabetic and oral glucose tolerance improving actions, particularly at the dose of 200 mg/kg in experimental animals. These activities of the plant extract may be related to the presence of secondary metabolites implicated in antidiabetic activities of plant extracts via different hepatic and extra-hepatic mechanisms. These results thus support the traditional use of the leaf extract for the management of diabetes mellitus.

Fast chromatographic separation for the quantitation of the main flavone dyes in Reseda luteola (weld).

In the past decades, there has been a renewed interest in the use of natural dye plants for textile dyeing, e.g. Reseda luteola (weld). Its main yellow dye constituents are the flavones luteolin-7,3'-O-diglucoside, luteolin-7-O-glucoside and luteolin. The aim of this work was to develop a simple validated industrially usable quantitative method to assess the flavone content of R. luteola samples. The flavones were overnight extracted from the dried and ground aerial parts of the plant at room temperature via maceration with methanol-water 8:2. Afterwards, they were quantified through internal standardisation against chrysin by RP-HPLC-UV at 345 nm. The efficiency of the one-step extraction was 95%. The limits of detection (LOD) and quantitation (LOQ) were ≤ 1 ng and ≤ 3 ng, respectively, providing ample sensitivity for the purpose. The precision expressed as relative standard deviation of the entire method was <6.5% for the combined content of luteolin-7,3'-O-diglucoside, luteolin-7-O-glucoside and luteolin. The average absolute recovery (accuracy) at three spiking levels was 102% (range: 98-107%) and the relative recovery ranged from 99 to 102%. The separation was initially carried out on a traditional 250 mm × 4.6 mm 5 µm HPLC column (80 min run time, 35.9 mL MeOH). It was then speeded up by the use of a 50 mm × 3.0mm 1.8 µm UHPLC column (5 min run time, 1.4 mL MeCN), while still using a conventional HPLC system. Whereas, the retention times on the UHPLC column were relatively less reproducible, cross-validation showed that the quantitation of luteolin-7,3'-O-diglucoside, luteolin-7-O-glucoside and luteolin was not statistically significantly different, with comparable precision. The method using the UHPLC column is more sensitive. The analytical method described meets the demand for a very small manpower input per sample and uses standard laboratory equipment. Usage of short UHPLC columns opens up interesting possibilities for modernising HPLC-based phytochemical analyses.

Effect of the heavy metals on developmental stages of ovule, pollen, and root proteins in Reseda lutea L. (Resedaceae).

Heavy metals are some of the most important environmental pollutants. Excessive amounts of heavy metals adversely affect plant growth and development. Also, the presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. The overall objective of this research was to elucidate some microscopic effects of heavy metals on the formation, development, and structure of pollen, ovule, and embryo and also root proteins in Reseda lutea L. For this purpose, the vicinity of Ahangaran lead-zinc mine (Hamedan, Iran) was chosen as a polluted area where amount of some heavy metals was several times higher than the natural soils. Flowers and young buds were collected from non-polluted and polluted plants, fixed in FAA(70), and studied during developmental stages by light microscopy. The results showed that heavy metals can cause some abnormalities during the pollen and ovule developmental process. The number of pollen grains was decreased, and their shape was changed. Increasing in thickness of the callosic wall and stabilizing of tapetum layer were observed in polluted plants. Asymmetrical formation of ovular integuments, degradation of egg apparatus, irregular formation of embryo sac, considerable vacuolation of embryonic cells, and degeneration of embryo in the late stage of heart-shaped embryo are the results of heavy metal pollution. For protein studies, young roots were harvested from plants exposed to pollution and non-exposed to pollution at the same time. Root proteins were extracted and studied by electrophoresis. The results revealed that some new proteins were synthesized in polluted samples that probably elevate plant tolerance to heavy metals.

Reseda luteola L. extract displays antiproliferative and pro-apoptotic activities that are related to its major flavonoids.

Reseda luteola L. has been used as a dye due to its high luteolin content since ancient times. However, no pharmacological studies have been performed with Reseda extracts so far. Here, we have assessed antiproliferative and apoptosis-inducing effects of the Reseda extract RF-40. It contains 40% flavonoids, primarily luteolin, but also luteolin-7-O-glucoside and apigenin. RF-40 and the isolated flavonoids dose-dependently inhibited cell proliferation and induced apoptotic oligonucleosomes in PHA-stimulated peripheral blood mononuclar cells. These effects were not due to cytotoxicity as shown with a luminometric ATP assay. Dose-response curves of RF-40 and the isolated flavonoids were similar, with luteolin being the most effective isolated flavonoid. Comparison of RF-40 to its major flavonoids revealed that the pharmacological effects of the extract can mostly be attributed to luteolin. We conclude that Reseda extract is an interesting raw material not only for dyeing purposes but also for further pharmacological investigation.

Topical application of solubilized Reseda luteola extract reduces ultraviolet B-induced inflammation in vivo.

We investigated the skin tolerance and anti-inflammatory potential of a nanoparticular solubilisate of a luteolin-rich Reseda extract (s-RE) in two independent studies in vivo. Reseda luteola extract containing 40% flavonoids was solubilized with polysorbate, resulting in product micelles with a diameter of 10 (+/-1.5)nm. Standardized inflammation was induced by irradiating test areas on the back of healthy volunteers with defined doses of ultraviolet B (UVB). In the first study different concentrations of s-RE were tested in 10 volunteers to evaluate dose-dependency of anti-inflammatory effects of s-RE. In the second randomized, double-blind, placebo-controlled study a defined concentration of s-RE (2.5%w/w) was tested in 40 volunteers in comparison to the vehicle (glycerol) and hydrocortisone (1%w/w). s-RE dose-dependently reduced UVB-induced erythema when applied 30 min before irradiation. To a lesser extent, topical application of s-RE after irradiation also reduced UVB-induced erythema. s-RE was as effective as hydrocortisone, whereas the vehicle had no effect. Occlusive application of s-RE on non-irradiated test sites did not cause any skin irritation. Due to excellent skin tolerance combined with potent anti-inflammatory properties s-RE bears potential especially for the prevention but also for the treatment of inflammatory skin conditions such as UV-induced erythema.

Characterization of weld (Reseda luteola L.) and spurge flax (Daphne gnidium L.) by high-performance liquid chromatography-diode array detection-mass spectrometry in Arraiolos historical textiles.

The natural dyes, and dye sources, in two seventeenth century Arraiolos carpets from the National Museum of Machado de Castro were analysed by high-performance liquid chromatography with UV-vis diode array detection (HPLC-DAD) and HPLC-mass spectrometry (LC-MS). Weld (Reseda luteola L.), indigo and spurge flax (Daphne gnidium L.) were found to be the dye sources, in agreement with original dyeing recipes collected during the nineteenth century. In order to fully characterize the plant sources, LC-MS conditions were optimized with plant extracts and the chromatographic separation and mass detection were enhanced. Extraction of the dyes, in the Arraiolos carpet samples, was performed using mild conditions that avoid glycoside decomposition. For the blues a dimethylformamide solution proved to be efficient for indigotin recovery. For all the other colours, an improved mild extraction method (with oxalic acid, methanol, acetone and water) was used, enabling to obtain the full dye source fingerprint, namely the flavonoid glycosides in the yellow dyes.

HPLC quantification of dye flavonoids in Reseda luteola L. from Portugal.

A HPLC method was developed for the simultaneous identification of Reseda luteola L. (weld) flavonoids and quantification of the main compounds responsible for the yellow color. This method was applied to a large number of wild Portuguese weld to evaluate its potential application as dyestuff for textile factories, as a substitute for the synthetic dyes currently used. Portuguese weld dyestuff content ranged between 1.04 and 5.87%, corresponding to a wide variation of the flavonoids amount (1.39-9.04%). Luteolin 4'-O-glucoside was found for the first time in R. luteola, but kaempferol, isorhamnetin, and their glycosides were not detected in the Portuguese specimens.

Acidichromism and ionochromism of luteolin and apigenin, the main components of the naturally occurring yellow weld: a spectrophotometric and fluorimetric study.

Luteolin and apigenin, extracted from Reseda luteola L., were spectrophotometrically and fluorimetrically studied. The spectra were investigated as a function of pH in methanol/water solutions (1/2, v/v) in the 2-12 pH range. The absorption spectra markedly shifted to the red by increasing the pH. Three acid-base dissociation steps were detected for luteolin (pK (a) = 6.9; 8.6; 10.3) and two for apigenin (pK (a) = 6.6; 9.3). Fluorescence emission was very weak or undetectable (Phi (F) < 10(-4)) in acidic solution, but increased in intensity with increasing the pH. Both molecules exhibited a great propensity towards complex formation with metal ions, with association constants on the order of 10(5)-10(7) for the first complexation step; in the presence of excess Al(3+) ions, multiple equilibria were detected. A marked fluorescence enhancement was observed upon complexation with Al(3+) ions (Phi (F) approximately 1 for luteolin and approximately 10(-2) for apigenin).