Extraction and analysis of free amino acids and 5'-nucleotides, the key contributors to the umami taste of seaweed.

Assessing the umami taste of seaweed on a chemical level can inform the use and selection of seaweed in European cuisine. Accordingly, we developed a method for the simultaneous extraction, separate clean-up and analysis of 21 free amino acids and 10 free nucleotides by reversed phase and mixed-mode HPLC respectively. Of multiple mouth emulating solvents, extracting in Milli-Q at 35 °C was found most suitable. This method showed good linearity (R2 > 0.9996), resolution (Rs ≥ 1.5) and picomole detection limits, and was successfully applied to determine the Equivalent Umami Concentration (EUC) and Taste Activity Values (TAV) of seven Dutch seaweed species. Phaeophyceae showed the highest EUC, followed by Chlorophyceae and Rhodophyceae (≈ 9.5, 3.7 and 1.1 g/100 g respectively). Glutamic acid always exceeded the TAV, while other umami compounds were species specific. Our method can accurately predict umami intensity and therefore contributes towards species selection for the European palette.

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.

Alizarin Grafting onto Ultrasmall ZnO Nanoparticles: Mode of Binding, Stability, and Colorant Studies.

The demand is rising for colorants that are obtained from natural resources, tolerant to industrial processing methods, and meet color quality demands. Herein, we report how relevant properties such as thermal stability and photostability of the natural colorant alizarin can be improved by grafting it onto ZnO nanoparticles (NPs), allowing application in a warm extrusion process for the fabrication of polyamide fibers. For this study, ZnO NPs (diameter 2.0 ± 0.6 nm) were synthesized and subsequently functionalized with alizarin. The alizarin-coated ZnO NPs (i.e., dyed nanoparticles, DNPs) were characterized. Thermogravimetric analysis and ultraviolet-visible (UV-vis) studies revealed that alizarin coating accounts for ∼65% (w/w) of the total mass of the DNPs. A subsequent detailed characterization with Fourier transform infrared (FT-IR), 1H nuclear magnetic resonance (NMR), 13C cross-polarization magic angle spinning (CP-MAS) NMR, X-ray photoelectron spectroscopy (XPS), and quantum chemistry studies using various density functional theory (DFT) functionals and basis sets indicated that binding onto the ZnO NPs occurred predominantly via the catechol moiety of alizarin. Importantly, this grafting increased the thermal stability of alizarin with >100 °C, which allowed the processing of the DNPs into polyamide fibers by warm extrusion at 260 °C. Evaluation of the lightfastness of the DNP-dyed nylon fibers revealed that the changes in color quantified via the distance metric ΔE* of alizarin when embedded in a hybrid material were 2.6-fold better compared to nylon fibers that were directly dyed with alizarin. This reveals that the process of immobilization of a natural dye onto ZnO nanoparticles indeed improves the dye properties significantly and opens the way for a wide range of further studies into surface-immobilized dyes.

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.

Two validated HPLC methods for the quantification of alizarin and other anthraquinones in Rubia tinctorum cultivars.

Direct and indirect HPLC-UV methods for the quantitative determination of anthraquinones in dried madder root have been developed, validated and compared. In the direct method, madder root was extracted twice with refluxing ethanol-water. This method allowed the determination of the two major native anthraquinone glycosides lucidin primeveroside and ruberythric acid. In the indirect extraction method, the anthraquinone glycosides were first converted into aglycones by endogenous enzymes and the aglycones were subsequently extracted with tetrahydrofuran-water and then analysed. In this case the anthraquinones alizarin, purpurin and nordamnacanthal may be determined. The content of nordamnacanthal is proportional to the amount of lucidin primeveroside originally present. The indirect extraction method is easier to apply. Different madder cultivars were screened for their anthraquinone content.

Chemical and enzymatic hydrolysis of anthraquinone glycosides from madder roots.

For the production of a commercially useful dye extract from madder, the glycoside ruberythric acid has to be hydrolysed to the aglycone alizarin which is the main dye component. An intrinsic problem is the simultaneous hydrolysis of the glycoside lucidin primeveroside to the unwanted mutagenic aglycone lucidin. Madder root was treated with strong acid, strong base or enzymes to convert ruberythric acid into alizarin and the anthraquinone compositions of the suspensions were analysed by HPLC. A cheap and easy method to hydrolyse ruberythric acid in madder root to alizarin without the formation of lucidin turned out to be the stirring of dried madder roots in water at room temperature for 90 min: this gave a suspension containing pseudopurpurin, munjistin, alizarin and nordamnacanthal. Native enzymes are responsible for the hydrolysis, after which lucidin is converted to nordamnacanthal by an endogenous oxidase.

Analysis of anthraquinones in Rubia tinctorum L. by liquid chromatography coupled with diode-array UV and mass spectrometric detection.

A liquid chromatographic (LC) method for the separation of both anthraquinone glycosides and aglycones in extracts of Rubia tinctorum was improved. For on-line MS detection atmospheric pressure chemical ionisation as well as electrospray ionisation (ESI) were used. The glycosides were ionised in both positive and negative ionisation (NI) mode, the aglycones only in the NI mode. With ESI ammonia was added to the eluent post-column to deprotonate the compounds. The efficiency of mass detection of the hydroxyanthraquinone aglycones was found to depend on the pKa value of the component. LC-diode-array detection and LC-MS provide useful complementary information for the identification of anthraquinones in plant extracts, which was proven with the identification of munjistin and pseudopurpurin.