Search Results (184)

Rouxiella badensis DSM 100043^T had been previously proven to produce a novel glucoselipid biosurfactant which has a very low critical micelle concentration (CMC) as well as very good stability against a wide range of pH, temperature, and salinity. In this study, we performed a function-based library screening from a R. badensis DSM 100043^T genome library to identify responsible genes for biosynthesis of this glucoselipid. The identified open reading frames (ORFs) were cloned into several constructs in Escherichia coli for gene permutation analysis and the individual products were analyzed using high-performance thin-layer chromatography (HPTLC). Products of interest from positive expression strains were purified and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) for further structure elucidation. Function-based screening of 5400 clones led to the identification of an operon containing three ORFs encoding acetyltransferase GlcA (ORF1), acyltransferase GlcB (ORF2), and phosphatase/HAD GlcC (ORF3). E. coli pCAT2, with all three ORFs, resulted in the production of identical R. badensis DSM 100043^T glucosedilipid with Glu-C_10:0-C_12:1 as the main congener. ORF2-deletion strain E. coli pAFP1 primarily produced glucosemonolipids, with Glu-C_10:0,3OH and Glu-C_12:0 as the major congeners, predominantly esterified at the C-2 position of the glucose moiety. Furthermore, fed-batch bioreactor cultivation of E. coli pCAT2 using glucose as the carbon source yielded a maximum glucosedilipid titer of 2.34 g/L after 25 h of fermentation, which is 55-fold higher than that produced by batch cultivation of R. badensis DSM 100043^T in the previous study. Full article

Celosiae Argentea Semen (CAS), derived from Celosia argentea L., is traditionally used in Korean and Chinese medicine to treat eye disorders and liver heat and is recognized in official Pharmacopeias. In contrast, Celosiae Cristatae Semen (CCS), despite its frequent presence in the market, is not officially listed. The morphological and chemical similarities between the two pose challenges for accurate identification. This study presents an integrative method combining digital image analysis and high-performance thin-layer chromatography coupled with mass spectrometry (HPTLC-MS) to differentiate CAS from CCS. Digital microscopy and ImageJ analysis showed that CCS has a projection area over twice that of CAS. Chemically, an optimized HPTLC method using ethyl acetate, methanol, water, and formic acid revealed distinct fingerprint patterns under UV 366 nm and white light. Notably, celosin F was exclusively detected in CAS, while celosin H, J, and K were characteristic of CCS. ESI-TOF-MS analysis confirmed these markers, resolving an overlap in R_F values. Repeatability tests showed total SDs of sucrose for intra-day, inter-day, and inter-analysis precision were 0.006, 0.004, and 0.005, respectively, confirming method reliability. This combined approach offers a rapid, reliable, and practical tool for distinguishing these two medicinal seeds, supporting enhanced quality control and regulatory standardization in pharmaceutical applications. Full article

Natural deep eutectic solvents (NADESs) have emerged as a promising eco-friendly alternative to petrochemicals for extracting plant metabolites. Considering that the demand for sustainable “green” ingredients for industrial applications is growing, those solvents are purported to develop extracts with interesting phytochemical fingerprints and biological activities. Given the interest in flavonoids from Chenopodium quinoa Willd. leaves, an efficient “green” extraction method was developed by investigating eight NADESs with defined molar ratios, i.e., malic acid-choline chloride (chcl)-water (w) (1:1:2, N1), chcl-glucose-w (5:2:5, N2), proline-malic acid-w (1:1:3, N3), glucose-fructose-sucrose-w (1:1:1:11, N4), 1,2-propanediol-chcl-w (1:1:1, N5), lactic acid-glucose-w (5:1:3, N6), glycerol-chcl-w (2:1:1, N7), and xylitol-chcl-w (1:2:3, N8). Rheological measurements of all NADESs confirmed their pseudoplastic behaviors. To improve the extraction processes, differential scanning calorimetry (DSC) allowed us to determine the maximum amount of water that could be added to the most stable NADES (N1, N2, N3, and N4; 17.5%, 20%, 10%, and 10% w/w, respectively) to lower their viscosities without disturbing their eutectic environments. The phytochemical compositions of NADES extracts were analyzed using high-performance thin-layer chromatography (HPTLC), and their free radical scavenging and α-amylase inhibitory properties were assessed using HPTLC-bioautography. N2, diluted with 20% of water, and N7 presented the best potential for replacing methanol for an eco-friendly extraction of flavonoids, radical scavengers, and α-amylase inhibitors from quinoa leaves. Their biological properties, combined with a good understanding of both thermal behavior and viscosity, make the obtained quinoa leaf NADES extracts good candidates for direct incorporation in nutraceutical formulations. Full article

Matcha, a finely powdered green tea, has been cherished in Japan for centuries, used in the traditional tea ceremony and nowadays also valued for its health-promoting properties. Cultivated under shaded conditions to enhance chlorophyll production, which gives the typical vibrant green color, matcha is rich in important bioactive compounds, including caffeine, catechins, and theanine. This study analyzes three matcha grades—ceremonial grade 1 (G1), grade 4 (G4), and food grade (FG)—to assess variations in their metabolite profiles. The Bligh–Dyer method was employed to extract polar and non-polar metabolites from organic and hydroalcoholic phases. High-performance thin-layer chromatography (HPTLC) was used for qualitative metabolite analysis, while nuclear magnetic resonance (NMR) spectroscopy was employed for both qualitative and quantitative analyses. Results reveal a decreasing gradient of amino acids and caffeine from grade 1 to food grade, while other metabolites, such as polyphenols, display an increasing trend. These findings suggest that factors such as harvesting time and leaf maturity significantly influence matcha’s chemical composition, providing a scientific basis for its quality differentiation and potential nutraceutical uses. Full article

Cannabis sativa L. is divided into three main groups: drug-type, intermediate-type, and fiber-type. The presence of tetrahydrocannabinol (THC) exceeding 0.2–0.3% in drug-type and intermediate Cannabis that utilized for recreational and medicinal purposes renders them illegal due to potential mental health implications. Fiber-type contains high cannabidiol (CBD) and low THC, making it suitable for household use such as textiles and animal feed. Accurate classification is essential to prevent misuse of the plant. High-performance thin-layer chromatography (HPTLC) and ultra-performance liquid chromatography (UPLC), used respectively for the qualitative and quantitative analyses of THC and CBD particularly in female inflorescences, categorized 85 samples of 46 cultivars used in this study into three distinct chemotypes. While chemotype analysis of a very specific organ of the plants accurately identifies Cannabis groups, it requires time-consuming plant development to maturity. Genotype analysis targeting tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes offers a faster alternative for classifying Cannabis types, allowing for sample determination from any part at any developmental stage of the plant. DNA sequencing allowed a phylogenetic analysis based on these genes, classifying all 85 samples of 46 cultivars into the same three groups identified by chemotype analysis. This study is the first to successfully examine the relationship between chemotype and genotype in 85 samples of 46 cultivars. Rapid identification of Cannabis types through genotype analysis lays the groundwork for future development of detection kits. Full article

Capparis spinosa is an edible plant with a long history in the Mediterranean region since antiquity. Its flower buds and leaves are mostly consumed salted or fermented (in vinegar) and are rarely eaten raw or dried. For the first time, caper samples subjected to different preservation processes (dried, salted, and desalted) were studied, foraged from the most producing Cycladic islands in Greece (Sifnos, Serifos, and Tinos). The quantitative determination of the flavonoids rutin and quercetin was carried out using high performance thin-layer chromatography (HPTLC), revealing the abundance of rutin in the buds and leaves (9.26–76.85 mg/g dry extract). Only one sample of desalted buds from Serifos showed a sufficient amount of quercetin (2.88 mg/g dry extract). The determination of total phenolic content (TPC) showed a decrease during brine (salted) preservation (11.7–37.7 mg GAE/g extract) compared to air-dried samples (50.9–62.4 mg GAE/g extract). The DPPH evaluation (8.0–35.2% inhibition at 200 μg/mL) was in agreement with the TPC results. All extracts showed stronger activity against Gram positive bacteria and the human pathogenic fungi C. glabrata. The samples from Sifnos exerted better bioactivities, with air-drying being the most effective preservation process in terms of antioxidant properties and phenolic content, although it resulted in a more bitter taste. Due to its high economic value, the caper holds great potential for further exploitation through better established and optimized processes in the food industry. Full article

The International Agency for Research on Cancer has studied and classified 1045 potential substances. It is therefore important to develop rapid screening methods to identify the mutagenicity of compounds and, further on, the intensity and number of individual mutagenic substances in complex sample mixtures. The current in vitro Ames assay in the microtiter plate format (MPF) uses a pH-sensitive detection as endpoint, however, acidic substances in complex mixtures may interfere the mutagenicity result. Hence, it was transferred to the planar assay format to be more selective for complex mixture testing. The co-culture of Salmonella Typhimurium strains TA98 and TA100 with an optical density of 0.4 at 600 nm was applied on a high-performance thin-layer chromatography silica gel 60 chromatogram and on-surface incubated for 5 h, which period was limited due to zone diffusion. Various positive controls were tested, and 4-nitrochinolin-N-oxide with a limit of detection of 100 ng was established as a positive control. However, due to the shorter incubation time, no mutagenic compounds were detectable or differentiable in the tested perfumes, herbal teas, margarines, and hand creams. This does not mean that the samples are mutagen-free, but it suggests that further improvements to the bioassay are urgently needed to increase the sensitivity and selectivity of the response. Compared to conventional sum value assays, a planar Ames assay performed on the separated and adsorbed sample components advances toxicology research because mutagenic compounds are separated from interfering molecules due to the integrated separation. It thus would provide a more selective detection of mutagens in complex mixtures and allow testing of large sample volumes or concentrated samples without matrix interference. Full article

This study aimed to examine wild-growing Hypericum perforatum L. tea (Hyperici herba) collected from Rtanj Mountain (Serbia). This research includes the following approaches: phytochemical and antioxidant characterization of H. perforatum infusion tea to determine its realistic composition (What do we consume when drinking the tea?), as well as a detailed examination of methanol(ic) extracts as the optimal extraction system. Due to the broad spectrum of both polar and nonpolar metabolites, 80% methanolic and pure methanol extracts were prepared for ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC Q-ToF) characterization through untargeted metabolomics analysis. Given the high diversity of compounds identified, the 80% methanolic extract was selected for further antioxidant examination and bioautographic characterization, including an antimicrobial activity assessment. UHPLC Q-ToF analysis identified 35 phenolics in the methanolic extract, compared to 25 metabolites in the infusion tea. The main differences were observed in flavonol/flavan-3-ol aglycones, xantones, and coumestans, which are more nonpolar compounds found only in the methanol(ic) system. Notably, specific H. perforatum metabolites were entirely absent in the infusion tea. Specifically, pseudohypericin, pseudoprotohypricin, and adhyperfirin were detected in the pure methanol extract, whereas hyperfirin was present in both methanol(ic) extracts. Additionally, eight furano-polycyclic polyprenylated acilphloroglucinols (FPPAPs) were identified in the methanol(ic) extracts as possible products of the thermal degradation and/or oxidation of hypericin/hyperforin. Both the infusion tea and methanolic extracts exhibited excellent antioxidant properties, with variations depending on the applied assay. High-performance thin-layer chromatography (HPTLC) analysis also confirmed the presence of a wide spectrum of phytochemical classes. Bioautography confirmed a promising activity of methanolic extracts against both Staphylococcus aureus and Klebsiella pneumoniae. Full article

The growing popularity and consumption of herbal slimming supplements can be attributed to their perception as natural products that lack side effects. However, the composition and ingredient quality listed on their labels often undergo insufficient control. As a result, some manufacturers add undeclared synthetic pharmaceuticals to enhance weight loss effects. The synthetic adulterants, particularly the anorectic stimulants, have been associated with increased risks of cardiovascular adverse effects, posing significant health risks to consumers. This study aimed to analyze various weight loss supplements marketed as “natural” products to detect possible adulterants. A new high-performance thin-layer chromatography (HPTLC) method was used for initial screening, while gas chromatography coupled with mass spectrometry (GC–MS) served as a confirmation tool. Additionally, high-performance liquid chromatography (HPLC) was employed to analyze phenolphthalein. A total of 34 supplements acquired online or from specialty stores were analyzed. It was found that most of them contain caffeine from herbal ingredients included in the products’ formulation. Some products list the added caffeine, but the measured levels significantly exceeded the labeled values. The most commonly detected adulterants were sibutramine and phenolphthalein. These results highlighted the inadequacies and inconsistencies in labeling, as all herbal supplements were declared “natural” despite containing adulterants. Furthermore, they highlighted the suitability of the HPTLC method as an effective and cost-effective screening tool for detecting adulterants in dietary supplements. Full article

Background/Objectives Galeopsis spp. (Lamiaceae) are widely distributed across extensive areas in Romania, being used mainly for their sedative, neuroprotective, antioxidant, anti-inflammatory, expectorant, astringent, and diuretic properties. The paper reports for the first time the investigation of the total phenolic content (TPC), total flavonoid content (TFC), and phenolic acid profile in the roots, aerial parts, and leaves from three wild-grown Galeopsis spp. (G. bifida Boenn., G. speciosa Mill., and G. tetrahit L.), along with their antioxidant and acetylcholinesterase (AChE) inhibitory potentials. Methods: The ultra-high-performance liquid chromatography/ultraviolet/mass spectrometry (HPLC/UV/MS) method was used for the identification and quantification of key phenolic acids. The spectrophotometric method was applied for the determination of TPC, TFC, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and also the ferric-reducing antioxidant power (FRAP). High-performance thin-layer chromatography (HPTLC) was employed for the assessment of in situ antioxidant (DPPH assay) and AChE inhibitory potentials. Results: Galeopsis spp. exhibit significant polyphenol accumulation. Chlorogenic acid was the most abundant compound, with the highest levels detected in G. tetrahit leaves (22,347.907 ± 1117.395 μg/g), followed by G. tetrahit aerial parts (11,678.509 ± 583.925 μg/g) and G. speciosa leaves (8712.628 ± 435.631 μg/g). G. tetrahit leaves had the highest DDPH radical scavenging activity, with a half-maximal inhibitory concentration (IC₅₀) of 0.458 ± 0.03 mg/mL, demonstrating a markedly stronger antioxidant effect. Leaves consistently showed the strongest DPPH activity across all species, with G. speciosa leaves also displaying a low IC₅₀ value of 0.789 ± 0.03 mg/mL, comparable to G. tetrahit. Aerial parts exhibited an intermediate effect, with G. bifida aerial parts showing an IC₅₀ of 8.102 ± 0.49 mg/mL, while G. tetrahit aerial parts demonstrated stronger activity at 1.511 ± 0.11 mg/mL. AChE inhibition activity increased progressively from the roots to aerial parts to leaves, with leaves consistently exhibiting the strongest inhibitory effects across all Galeopsis spp. G. tetrahit leaves had the strongest inhibition, with an IC₅₀ of 4.002 ± 0.32 mg/mL, followed by G. speciosa leaves (6.92 ± 0.14 mg/mL) and G. bifida leaves (6.97 ± 0.68 mg/mL). Conclusions: Our study provides a comprehensive analysis of the phenolic acid content, in vitro antioxidant activity, and neuroprotective potential of three Galeopsis spp. (G. bifida, G. speciosa, and G. tetrahit) from the southwestern Romanian flora. Full article

Background: GM3 Synthase Deficiency (GM3SD) is a rare autosomal recessive neurodevelopmental disease characterized by recurrent seizures and neurological deficits. The disorder stems from mutations in the ST3GAL5 gene, encoding GM3 synthase (GM3S), a key enzyme in ganglioside biosynthesis. While enzyme deficiencies affecting ganglioside catabolism are well-documented, the consequences of impaired ganglioside biosynthesis remain less explored. Methods: To investigate GM3SD, we used a Human Embryonic Kidney 293-T (HEK293-T) knockout (KO) cell model generated via CRISPR/Cas9 technology. Lipid composition was assessed via high-performance thin-layer chromatography (HPTLC); glycohydrolase activity in lysosomal and plasma membrane (PM) fractions was enzymatically analyzed. Lysosomal homeostasis was evaluated through protein content analysis and immunofluorescence, and cellular bioenergetics was measured using a luminescence-based assay. Results: Lipidome profiling revealed a significant accumulation of lactosylceramide (LacCer), the substrate of GM3S, along with increased levels of monosialyl-globoside Gb5 (MSGb5), indicating a metabolic shift in glycosphingolipid biosynthesis. Lipid raft analysis revealed elevated cholesterol levels, which may impair microdomain fluidity and signal transduction. Furthermore, altered activity of lysosomal and plasma membrane (PM)-associated glycohydrolases suggests secondary deregulation of glycosphingolipid metabolism, potentially contributing to abnormal lipid patterns. In addition, we observed increased lysosomal mass, indicating potential lysosomal homeostasis dysregulation. Finally, decreased adenosine triphosphate (ATP) levels point to impaired cellular bioenergetics, emphasizing the metabolic consequences of GM3SD. Conclusions: Together, these findings provide novel insights into the molecular alterations associated with GM3SD and establish the HEK293-T KO model as a promising platform for evaluating potential therapeutic strategies. Full article

Chaga (Inonotus obliquus) is an increasingly used natural product in botanical dietary supplements, valued for its bioactive compounds. However, inconsistent standardized analytical methods raise concerns over product authenticity, mislabeling, and quality control. This study employs a multi-analytical approach to differentiate wildcrafted chaga canker from North American chaga dietary supplements, particularly those containing mycelia fermented grain products. High-Performance Thin-Layer Chromatography (HPTLC), Liquid Chromatography with Evaporative Light Scattering Detection (LC-ELSD) or Photo/Diode Array Detection (LC-PDA/DAD), Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS), Nuclear Magnetic Resonance (NMR) spectroscopy, UV-Vis spectrophotometry, and iodine-starch assays were used to evaluate key markers, including triterpenoids, polysaccharides, and melanin. Whole chaga canker contained triterpenoids (inotodiol, trametenolic acid) and phenolics, like osmundacetone, while melanin absorbance at 500 nm differentiated it from fermented grain products. β-Glucan quantification and iodine-starch assays confirmed starch-rich composition in fermented grains and its absence in authentic chaga canker. NMR fingerprinting and LC-QToF-MS metabolomics demonstrated stark compositional deviations between wildcrafted chaga canker, I. obliquus mycelium, and fermented grain products. By integrating complementary techniques, we establish a framework that can reliably distinguish genuine chaga canker from misrepresented products, ensuring consumer safety and fostering trust in the functional mushroom, canker, and mycelium markets. Full article

Frankincense resin (Boswellia serrata), native to arid regions of India, the Middle East, and parts of Africa, has been highly valued for its medicinal properties. This study evaluated the antimicrobial potential of methanolic extracts of Boswellia serrata resin against Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. High-performance thin-layer chromatography (HPTLC) coupled with bioautography identified bioactive zones, while Liquid Chromatography–Mass Spectrometry (LC-MS) quantified the phenolic and terpenoid compounds. The cytotoxicity was assessed on HaCaT human keratinocyte cells to evaluate the safety for dermatological applications. The results demonstrated significant antibacterial activity, particularly against S. aureus and L. monocytogenes. The bioautograms revealed that samples from central and southern Serbia showed the highest antimicrobial effect against the tested bacterial strains. The active compounds included 11-keto-β-boswellic acid (up to 3733.96 μg/g), gallic acid (110.93 μg/g), and naringenin (53.13 μg/g). Cytotoxicity assays confirmed non-toxic effects at 10 µg/mL, with sample 6 enhancing the keratinocyte viability by 137%, while higher concentrations (50 µg/mL) showed variable cytotoxicity. These findings highlight the potential of B. serrata resin as a natural antimicrobial agent, particularly against antibiotic-resistant pathogens. Its therapeutic applicability in pharmaceutical and cosmetic formulations is promising provided that dosing ensures a balance between efficacy and safety. Full article

This study investigates the physiochemical properties, chemical composition, and antioxidant activity of Australian stingless bee honey blends from two bee species, Tetragonula carbonaria and Tetragonula hockingsi, harvested in Burpengary East, Queensland at different times of the year. The moisture content of the honey samples ranged from 26.5% to 30.0%, total soluble solids from 70.0 to 73.5° Brix, and pH from 3.57 to 4.19. The main sugars identified were trehalulose (13.9 to 30.3 g/100 g), fructose (12.9 to 32.3 g/100 g), and glucose (4.80 to 23.7 g/100 g). The total phenolic content (TPC), measured using the Folin–Ciocalteu assay, ranged from 26.1 to 58.6 mg of gallic acid equivalents/100 g. The antioxidant activity was investigated with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, with values ranging from 1.39 to 6.08 mmol of Trolox equivalents/kg. Antioxidant constituents were determined using a High-Performance Thin-Layer Chromatography (HPTLC)-DPPH assay. The HPTLC-DPPH analysis revealed that honey samples collected in May 2022 contained the highest number of antioxidant compounds. Some constituents were identified using an HPTLC-derived database and also quantified utilising HPTLC analysis. Lumichrome was present in all honey samples, while luteolin and kaempferide were detected only in some. Kaempferol or isorhamnetin was also found to be present, although a definitive distinction between these two chemically closely related compounds could not be made by HPTLC analysis. The results showed that honey produced by Tetragonula hockingsi and Tetragonula carbonaria shares similar properties and composition when harvested at the same time, with only minor differences in moisture, fructose, and glucose content. Full article

A simple and robust two-dimensional chromatographic fractionation protocol for the isolation of the neuroprotective compound erinacine A from Hericium erinaceus is proposed. This production platform yielded 19.4 mg of erinacine A from approximately 130 g of mushroom material, with a chromatographic purity of 97.4%. The procedure includes extraction, concentration, fractionation, purification, and characterisation of the bioactive compound. The crude H. erinaceus extract was fractionated in the first dimension by normal-phase flash chromatography, and the fraction containing erinacine A was further purified in the second dimension by semi-preparative reversed-phase chromatography. This strategy utilises the orthogonality of the two chromatographic modes to effectively eliminate difficult impurities, including structural isomers and analogues of erinacine A. Complementary analytical approaches such as high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography with ultraviolet and tandem mass spectrometric detection (HPLC–UV–MS/MS) were employed to unambiguously confirm erinacine A in the isolated fractions, while HPLC with a charged aerosol detector (CAD) was used to determine its purity. Given the limited commercial availability and the high price of erinacine A, the described method offers a straightforward and cost-effective alternative to obtain this valuable compound for further research and applications. Full article