Column Chromatography Research Articles

Isolation and Identification of Monoterpenoids from Callicarpa Nudiflora.

A phytochemical and directional investigation was carried out on the leaves of Callicarpa nudiflora. Various separation methods including silica gel, sephadex LH-20, medium pressure liquid chromatography (MPLC), preparative thin-layer chromatography (p-TLC) and preparative high performance liquid chromatography (p-HPLC) were used to isolate 10 compounds. The comprehensive structural elucidation of five novel monoterpenoids (1-5) was achieved through multi-spectroscopic analyses, and advanced quantum mechanical electronic circular dichroism (ECD) calculations. Furthermore, the anti-inflammatory activity of the identified compounds was assessed.

Novel Antioxidant Peptides from Fermented Whey Protein by Lactobacillus rhamnosus B2-1: Separation and Identification by in Vitro and in Silico Approaches.

Whey is a byproduct of the dairy industry and is rich in protein. To enhance the significance of such byproducts and find efficacious antioxidants for combating oxidative stress, this study reported on the preparation, purification, and identification of novel peptides with antioxidant activities from whey protein metabolites following fermentation by Lactobacillus rhamnosus B2-1. The isolation and identification processes involved macroporous adsorption resin column chromatography, gel filtration column chromatography, and liquid chromatography-tandem mass spectrometry. Therein, three novel antioxidant peptides (PKYPVEPF, LEASPEVI, and YPFPGPIHNS) were selected to be synthesized, and they demonstrated remarkable antioxidant activities in vitro chemical assays. PKYPVEPF, LEASPEVI, and YPFPGPIHNS (100 μg/mL) displayed a notable cytoprotective impact on HepG2 cells under oxidative stress induced by H2O2, increasing the cell viability from 49.02 ± 3.05% to 88.59 ± 10.49%, 82.38 ± 19.16%, and 85.15 ± 7.19%, respectively. Moreover, the peptides boosted the activities of catalase and superoxide dismutase in damaged cells and reduced reactive oxygen species levels. The molecular docking studies highlighted that these antioxidant peptides efficiently bound to key amino acids in the Kelch domain of Keap1, thereby preventing the interaction between Keap1 and Nrf2. In conclusion, PKYPVEPF, LEASPEVI, and YPFPGPIHNS demonstrated substantial antioxidant activity, suggesting their potential for widespread application as functional food additives and ingredients.

HPLC analysis and antimicrobial activity of Vitex altissima leaves extracts

The exploration of ethnomedicinal plants for phytochemical and pharmacological properties is crucial for developing novel therapeutics for chronic diseases. Vitex altissima, known as the Peacock chaste tree, is a significant member of the Verbenaceae family, is widely utilized in traditional medicine. This study investigates the phytochemical composition and antibacterial properties of V. altissima, a plant recognized for its medicinal use by various indigenous communities, including the Malayali tribes of Servarayan hills. The study involved extracting phytochemicals from the leaves of V. altissima using hexane, ethyl acetate and methanol through soxhlet extraction. The extracts were analyzed for secondary metabolites, including carbohydrates, proteins, amino acids, steroids, glycosides, alkaloids, tannins, phenolics, flavonoids, terpenoids, saponins, anthraquinones, oils and resins, diterpenes, phlobatannins and coumarins. Quantitative assessments of phenolic compounds, flavonoids and alkaloids were performed. Column chromatography was employed to fractionate the methanolic extract, resulting in 12 distinct fractions. These fractions were screened for antibacterial activity against E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aureus using the agar well diffusion method. The minimal inhibitory concentration (MIC) was determined through a 2-fold serial dilution technique. High-performance liquid chromatography (HPLC) was utilized to identify the polyphenolic compounds responsible for antibacterial activity. Phytochemical screening revealed a diverse array of bioactive compounds in the extracts, with methanol extracts showing the highest total phenolic content (218 ± 7.21 mg GAE/g of extract) and significant antibacterial activity. The ethyl acetate extract exhibited the highest total flavonoid content (40.67 ± 6.65 mg QE/g of extract). Among the fractions, the column fraction from the chloroform-methanol gradient (VACF) demonstrated superior antimicrobial activity, particularly against S. mutans and P. aureginosa. The MIC values for VACF were 427 µg/mL for P. aeruginosa and 400 µg/mL for S. aureus, indicating potent antibacterial properties. HPLC analysis identified key polyphenolic compounds, including p-coumaric acid, ferulic acid and elagic acid, as the primary contributors to the antibacterial activity. The presence of these compounds aligns with the observed antimicrobial efficacy and highlights the potential of V. altissima extracts as a source of natural antibacterial agents.

Structural characteristics of a purified Evodiae fructus polysaccharide and its gastroprotection and relevant mechanism against alcohol-induced gastric lesions in rats

Evodiae fructus polysaccharide (EFP) has been previously shown to protect against alcohol-induced gastric lesions. However, which and how active fractions in EFP exert gastroprotection remains unclear. This study aimed to characterize the structure of the purified fraction (EFP-2-1) of EFP, and investigate its gastroprotection and underlying mechanisms. EFP-2-1 was obtained through column chromatography, and was characterized using instrumental analytical techniques. Gastroprotective effect of EFP-2-1 was evaluated using alcohol-induced gastric lesions in rats, and its mechanism was explored through proteomics, metabolomics and diversity sequencing. Results showed that EFP-2-1 had a molecular weight of 7.3 kDa, and consisted mainly of rhamnose, galacturonic acid, galactose and arabinose. Its backbone contained HG and RG-I domains, and branched with →5)-α-l-Araf-(1→, α-l-Araf-(1→ and →4)-β-d-Galp-(1→ residues. EFP-2-1 reduced gastric lesions and the levels of MDA, TNF-α and IL-6, activated PPARγ, primarily altered protein digestion and absorption and bile secretion metabolic pathways, regulated gut microbiota like Faecalibaculum and Lachnoclostridium, and increased short-chain fatty acids production. Correlations were observed among the gut microbiota, metabolites and biochemical indexes influenced by EFP-2-1. These findings suggest that EFP-2-1 is an active fraction of EFP for protecting against alcohol-induced gastric lesions, which may be linked to PPARγ activation, gut microbiota and serum metabolism.

Revealing the backbone structures of land-based fulvic acids derived from river and soil through n-butylsilane reduction.

While ultra-high-resolution mass spectrometry has enabled the identification of the molecular composition of dissolved organic matter (DOM), elucidating its molecular structure remains a challenging endeavor. Here, two fulvic acids (FAs), one from river and the other from forest soil, were subjected to reduction using an optimized n-butylsilane (n-BS) reduction method. The reduction products were purified through a combination of liquid-liquid extraction and silica gel column chromatography, resulting in the separation into saturates, aromatics, and polar products. The polar products were analyzed by high-resolution mass spectrometry (HRMS), and the saturates and aromatics were analyzed using gas chromatography-mass spectrometry (GC-MS). HRMS results showed that the number of oxygen atoms and double-bond equivalent (DBE) values of FA decreased after reduction. GC-MS results revealed that a total of 270 hydrocarbon monomers were identified from the reduction products of a single sample, with the highest carbon number of cycloalkanes reaching C33. For the first time, steranes and hopanes were detected in the reduction products, potentially serving as evidence for the existence of carboxyl-rich alicyclic molecule (CRAM) precursors. Additionally, a significant number of polycyclic aromatic hydrocarbons were identified, and the potential sources of various compounds were preliminarily inferred based on their isomers. This study extends the knowledge of the possible backbone structure of the DOM and provides a new potential tool for investigating the origin and transformation mechanisms of DOM.

Identification and in vitro genotoxicity assessment of forced degradation products of glimepiride and glyburide using HEK cell-based COMET assay.

This study focuses on characterizing the forced degradation products of antidiabetic drugs glimepiride (GMD) and glyburide (GBD), with previously unexplored genotoxicity. Drugs underwent stress induced by acid, base, and hydrogen peroxide. For GMD, impurities were profiled and isolated using Hypersil Gold C8 (250 × 10 mm, 5μ) through semi-preparative HPLC with a fraction collector. For GBD, impurity profiling was performed using semi-preparative HPLC (Hypersil GOLD C18, 250 × 10 mm, 5μ), and reverse-phase flash chromatography (FP ECOFLEX C18 4g column) for isolation. Although five GMD and three GBD impurities were detected, only three GMD and two GBD impurities were separated and assessed for purity using analytical RP-HPLC with the purity percentages ranging from 96.6% to 99.9%. LC-Orbitrap MS was used to identify these three GMD impurities (m/z: 408.122, 338.340, 381.160) and two GBD impurities (m/z: 369.065, 325.283). ProTox-II in silico predictions classified all impurities as class 4 and 5, with no positive genotoxicity indications. In vitro comet assays, using HEK cells, indicated that for GMD, impurity 2 and impurity 5 were less genotoxic, whereas impurity 4 exhibited genotoxicity. For GBD, both impurities 1 and 3 were found to be genotoxic, with impurity 3 showing a higher level of genotoxicity than impurity 1.

Structural characterization of a cell wall-associated polysaccharide of Nocardia rubra FIM-21 and its immunostimulatory activity

In this work, a neutral polysaccharide named NP-1 was obtained from the cell wall of Nocardia rubra FIM-21 by column chromatography. Its chemical structure was characterized by methylation analysis, 2D NMR spectroscopic study, and other techniques. It was shown that NP-1 contained five types of monosacchrides, including mannose (Man), glucose (Glc), N-acetylglucosamine (GlcpNAc), galactose (Gal) and arabinose (Ara) in a molar ratio of 7.7:3:2:1:1.5. According to linkage analysis and NMR spectra, the backbone contains 1,6-linked Manp, 1,4-linked Glcp, and 1,6-linked Galp, with branches substituted at the C-6 of 1,4-linked Glcp and the C-2 of 1,6-linked Galp by GlcpNAc, and at C-2 of 1,6-linked Manp by Araf, Araf-1,5-Araf or GlcpNAc. Biological activity research showed that NP-1 could stimulate the proliferation of RAW264.7 cells and enhance their phagocytosis and release of NO.

Sustainable methods for producing food-derived bioactive compounds

Background: Pursuing alternative starting materials has gained significant importance in the food and drug industries, driven by the global shift towards sustainable and renewable resources. Terpenes, naturally occurring hydrocarbons derived from plants, present a promising solution due to their abundant availability and range of physiological activities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. Objective: This study aims to develop a waste-free synthesis methodology for functionalizing limonene and myrcene, two notable terpenes, through Atom Transfer Radical Reactions (ATRR). The objective is to achieve high yields and purities while enhancing the therapeutic potential of these compounds and adhering to green chemistry principles. Methods: A catalytic complex of Cu(I)Br and dimethyl sulfoxide (DMSO) was used to facilitate the coupling of limonene and myrcene with various trichloroacetic acid derivatives. Reactions were performed at 80°C for 90 minutes, and products were purified by column chromatography. The synthesized compounds were characterized using Gas Chromatography-Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance (NMR). Microbiological analyses were conducted using the disk-diffusion method, and statistical analyses were performed to assess the reliability of the experiments. Results: The ATRR methodology enabled the efficient functionalization of limonene and myrcene, achieving high yields and purities. The best yield for limonene was 70%, and for myrcene, 82%, both using trichloroacetonitrile. The Cu(I) catalytic system showed dualistic biological effects, with 3% concentration exhibiting strong bactericidal and fungicidal activity, while 0.03% promoted growth. These results demonstrate the potential of the methodology and the catalytic system for bioactive compound synthesis and agricultural applications. Conclusion: This study demonstrates the viability of ATRR for efficiently functionalizing limonene and myrcene, achieving high yields and purities. The synthesized compounds show promise for pharmaceutical development. The dual antibacterial and antifungal activities of the Cu(I) catalytic system also suggest potential applications in wastewater reutilization. Future work should explore the biological effects and environmental impact of this process. Keywords: Terpenes, ATR reactions, Green Chemistry, Bioactive compounds, Therapeutic potential.

Introducing Column Chromatography through Colorful Reactions of Guaiazulene

Introducing Column Chromatography through Colorful Reactions of Guaiazulene

Consolidated Curium Reprocessing Procedure Inspires Molecular Design for Sensitized Curium Circularly Polarized Luminescence.

Curium's stable redox chemistry and ability to emit strong metal-based luminescence make it uniquely suitable for spectroscopic studies among the actinide series. Targeted ligand and coordination compound design can support both fundamental electronic structure studies and industrial safeguards with the identification of unique spectroscopic signatures. However, limited availability, inherent radioactive hazards, and arduous purifications have long inhibited such investigations of this element. A consolidated reprocessing procedure for curium has been developed for the milligram scale. The recovery of not only standard legacy curium samples but also hazardous legacy perchlorate containing curium samples was achieved, culminating in column chromatography utilizing the extraction resin DGA (N,N,N',N'-tetra-2-ethylhexyldiglycolamide, branched). Surprisingly, controlled elution of the Cm band from the extraction resin was followed through bright pink luminescence triggered by an inexpensive hand-held UV-vis lamp (380-400 nm). This observation inspired the design of an enantiopure, C2-symmetrical ligand bearing a chiral (trans-1,2-diaminocyclohexane) backbone with achiral DGA moieties (N,N,N',N'-tetra-n-octylacetamide), that enabled rarely observed curium circularly polarized luminescence upon metal chelation. These combined achievements should unlock more luminescence and circularly polarized luminescence studies of curium, and enable the recovery of many curium and other trivalent actinide samples.

Encapsulation of Phloroglucinol from Rosenvingea intricata Macroalgae with Zinc Oxide Nanoparticles against A549 Lung Cancer Cells

Background/Objectives: Phloroglucinol (PHL), a phenolic compound extracted from the brown alga Rosenvingea intricata, exhibits potent antioxidant and anticancer properties. This study aims to extract, purify, and characterize PHL, and further develop functionalized zinc oxide nanoparticles (ZnO NPs) loaded with PHL to enhance its therapeutic potential. Methods: PHL was extracted using acetone and purified through Sephadex LH-20 column chromatography, yielding a highly enriched fraction (F-3). The purified compound was characterized by FTIR, HPLC, NMR, and LC-MS. ZnO NPs were synthesized, PEGylated, and conjugated with PHL, forming ZnO-PEG-PHL NPs. Their characterization included DLS, zeta potential, XRD, SEM-EDAX, and encapsulation efficiency studies. Antioxidant assays (DPPH, FRAP, ABTS, RPA) were performed and in vitro cytotoxicity on A549 lung cancer cells were determined to evaluate the therapeutic efficacy of PHL. Results: The purified PHL fraction showed a high phenolic content (45.65 PHL mg/g), which was was confirmed by spectral analysis. The ZnO-PEG-PHL NPs increased in size from 32.36 nm to 46.68 nm, with their zeta potential shifting from −37.87 mV to −26.82 mV. The antioxidant activity was superior for the ZnO-PEG-PHL NPs in all assays, while the in vitro cytotoxicity tests showed an IC50 of 40 µg/mL compared to 60 µg/mL for the ZnO NPs and 70 µg/mL for PHL. Apoptotic studies revealed significant cell cycle arrest and apoptosis induction. Conclusions: The synthesized ZnO-PEG-PHL NPs demonstrated enhanced antioxidant and anticancer properties, making them promising candidates for cancer therapy and antioxidant applications.

Pentacyclic triterpenes as bioactive compounds isolated from Mauritia flexuosa L. f. acting against the Alzheimer’s disease

Alzheimer’s disease is a significant concern due to its high prevalence and the limitations of current treatments. In our research, we investigated Mauritia flexuosa, a medicinal plant traditionally used for headaches, to identify active compounds with potential anti-Alzheimer’s effects. Three pentacyclic triterpenes were isolated through column chromatography and characterised from the dichloromethane/methanol extract from Mauritia flexuosa (DCMEMf), with (3β)-3-hydroxy-11-oxours-12-en-28-oic acid (3) showing the highest in vitro activity in the HMC3 and SVG p12 cell lines. Compound 3 inhibited the pharmacological targets NF-κB, PGE2, IDO1, and EGFR with IC50 values of 9.83, 3.86, 1.63 μM, and 49.57 nM, respectively, attributed to a hydroxyl group at the C-3 position of its structure. These findings suggest the potential of these compounds in treating neurological diseases, including headaches, and offer promising prospects for the development of new therapies against Alzheimer’s.

In vitro and silico Analysis of Amentoflavone against Candida albicans with a Competitive Action about Amphotericin B

Introduction: Antifungal resistance is a global health problem, and alternatives for control and treatment must be sought. Thus, substances isolated from plants with antifungal capacity have received considerable attention in the pharmaceutical sector. Ouratea fieldingiana, popularly known as batiputá, is a tree species found in northeastern Brazil, with its leaves rich in biflavonoids such as amentoflavone. Biflavonoids are well-studied due to their high antifungal capacity. Objectives: Carry out the isolation, identification, and characterization of the compound amentoflavone (AMT), as well as evaluate its antifungal activity and modulatory effect against strains of Candida albicans, also including the computational study of the mechanism of action of the compound AMT against the Als3 and Sap5 enzymes of C. albicans. Methodology: Column chromatography was used to isolate the AMT compound from the species O. fieldingiana, and its characterization was performed by high-performance liquid chromatography (HPLC). The antifungal activity was analyzed by the broth microdilution method. Modulatory activity assays were performed by the checkerboard technique using AMT and amphotericin B (AmB) as a standard. For the in silico study, computational simulations of the interaction between the enzymes Als3 and Sap5, and AMT were performed using the Vina AutoDock Code. Results: The AMT compound showed an inhibitory effect against all strains of C. albicans. Regarding modulatory activity, an indifferent and antagonistic effect was observed for all C. albicans strains tested. Molecular docking with AMT showed higher affinity energy for the Als3 and Sap5 enzymes than AmB. The results obtained in this study suggest that AMT has antifungal effects, as well as a high affinity for the Als3 and Sap5 enzymes of C. albicans. Conclusion: These results prove that the AMT compound could be a potential source of a new biotechnological product, acting as a natural antifungal agent.

A-320 Determination of Guanidinoacetate Methyltransferase Enzyme Biomarkers in Newborn Dried Blood Spot by Tandem Mass Spectrometry-A Two Tier Approach

Abstract Background Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Following a recommendation by the Recommended Uniform Screening Panel and in line with California State statute, all California newborns will be screened for GAMT deficiency beginning July 2024. Here, we developed and validated a quantitative two-tier tandem mass spectrometry (MS/MS) assay for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). Methods This method punches a 3.2 mm disc of controls and newborn patient DBS into a 96-well plate. 100 μL of extraction solution, containing internal standards of GAMT biomarkers, is added to each well and incubated at 40°C for 45 minutes. The extract is transferred, evaporated, and derivatized into a 3N-butanol-HCl solution at 60°C for 30 minutes. The excess HCl is evaporated to dryness. The conversion of GUAC and creatine to their butyl esters has improved their detection sensitivity by MS/MS. The butyl esters are reconstituted into the mobile phase and shaken for 10 minutes at 27°C. The final 10 μL extract was analyzed by flow injection analysis (FIA)-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. The presumptive positive samples from initial analysis are prepared following the same method, and a 5 μL extract is resolved through an ultra-performance liquid chromatography (UPLC) column and analyzed by UPLC-MS/MS in MRM positive ion mode with a total run time of 3.5 minutes per sample. The validation was performed by assessing the linearity, accuracy, imprecision, carryover, limit of detection (LOD) and lower limit of quantitation (LLOQ), matrix effect, analytical measurement range (AMR), percent total allowable error (TEa), and reference range. Results This method is linear for GUAC, creatine and creatinine over a measured range of 1.17-40.00, 121.20-857.36 and 41.34-387.46 µmol/L respectively. The R2 values=1.000, 0.997, 0.9998; slope=0.999, 0.9945, 0.9982; intercept=0.0398, 4.881, 1.7027; mean recoveries=99-104%, 100-104%, 100-104%; intra-assay precision (n=20) =4.05-5.95%, 4.62-5.31%, 3.27-5.19%, inter-assay precision (n=40)=3.7-12.6%, 4.3-7.6%, 3.8-7.4%; TEa=6-21%, 7-17%, 6-17%; LOD=0.12, 0.54, 1.11 µmol/L; AMR=0.33-1300, 1.11-2700, 3.50-4500 µmol/L; median patient results observed from 2293 normal patient=1.48, 494.02, 89.97 μmol/L for GUAC, creatine, and creatinine respectively. The tier-1 and tier-2 assay screening cutoff for GUAC at the population 95th percentile is 2.53 and 4.55 μmol/L respectively. Conclusions The tier-1 and tier-2 methods has been developed and validated to determine GAMT biomarkers in newborn DBS, making it suitable for routine screening of ∼2000 newborn specimens per day by tier-1 and followed by more specific tier-2 to separate GAMT biomarkers from potential isobaric interfering compounds. This two-tier approach greatly improved the test performance and significantly reduced false positive rate.

Anti-Melanogenic Effects of a Polysaccharide Isolated from Undaria pinnatifida Sporophyll Extracts

Undaria pinnatifida is a temperate brown alga known to exert free radical-scavenging and anti-inflammatory effects. In this study, we investigated the skin-whitening effects of U. pinnatifida sporophyll extracts (UPEs) in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. The crude polysaccharide fraction (UPF) was obtained via ethanol precipitation. Four polysaccharide fractions (UPF1–4) were isolated and purified using ion-exchange column chromatography, and their anti-melanogenic activity was evaluated. UPF3 exhibited the highest anti-melanogenic activity, showing the highest sulfate (39.79%), fucose (143 μg/mg), and galactose (208 μg/mg) contents. UPF3 significantly inhibited intracellular tyrosinase activity in B16F10 cells. We also evaluated the melanogenic signaling pathway to determine the mechanism of action of UPF3 in melanongenesis. UPF3 reduced the expression of tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and tyrosinase, which play important roles in melanin production. Therefore, UPF3 has high potential for use in skin-whitening functional pharmaceuticals and cosmetics.

B-292 Detection of natural toxins derived from potatoes by biotin-streptavidin direct ELISA

Abstract Background α-solanine (SO) and α-chaconine (CHA) are natural toxins of potato and often cause food poisoning worldwide. We have previously developed a polyclonal antibody (anti-Sold antibody) that bind to SO and CHA and used it in an enzyme-linked immunosorbent assay (ELISA). Our previously constructed ELISA (conventional ELISA) has a weak point of low sensitivity in detecting SO and CHA, we have tried to improve it in this study. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Anti-Sold antibody was then purified from rabbit serum using affinity column chromatography. In our ELISA method, named “B-S direct ELISA”, biotin-labeled anti-Sold antibody is bound to SO and CHA in the sample, followed by the binding of peroxidase-labeled streptavidin. To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results The absorbance of the samples containing SO and CHA prepared in 10 mM phosphate buffer saline (PBS) was about 5-fold higher than that of the conventional ELISA. Furthermore, the absorbance of the samples containing SO and CHA prepared in human serum and urine was enhanced about 2.5- and 1.6-fold, respectively, compared to that of the conventional ELISA. The limit of detection in B-S direct ELISA was 0.32 ng/mL in 10 mM PBS, 2.08 ng/mL in serum, and 4.88 ng/mL in urine, respectively. Using this ELISA, it was also possible to measure the content of SO and CHA in extracts of potato tubers, peels, and sprouts. Both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.4- and 2.2-fold higher, respectively, in each part than those in the extracts from fresh potatoes. Furthermore, the potato sprout extract contained approximately 50-fold more SO and CHA than the tuber and 9-fold more than the peel. On the other hand, B-S direct ELISA cross-reacted with solanidine and solasodine, which have similar chemical structures to SO and CHA, and also with cholesterol and vitamin D3 in serum. Conclusions Based on our results, B-S direct ELISA presented greater performance in detecting SO and CHA than our conventional ELISA. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although this ELISA has a detection performance applicable to human biological samples, cross-reactivity should be fully considered when judging the results.

Determination of multiple restricted and prohibited ingredients in hair dyes using a novel 3D stationary phase coupled with gas chromatography-mass spectrometry

Hair dyes (HDs) are mainly composed of various benzene series, amines, and phenolic compounds. These ingredients are well known to have allergenic, teratogenic, and carcinogenic properties. As such, the presence of these ingredients in HDs has received increased attention in recent years. At present, the applications of traditional analytical and detection methods and commercial chromatographic columns are limited by problems such as poor qualitative analysis and inaccurate quantification. Thus, the development of new analytical and detection technologies and stationary phases is an urgent endeavor. Moreover, HDs contain complex compounds and exhibit significant matrix interference. Hence, appropriate sample pretreatment methods are necessary to analyze HDs. In this study, the 3D nonpolar rigid structure of triptycene (TP) was combined with the polar flexible chains of polyethylene glycol (PEG) to design and synthesize a TP derivative, TP-PEG, as a stationary phase for chromatographic columns. The stationary phase enabled the expansion of the selection range for polar and nonpolar analytes. Subsequently, gas chromatography-mass spectrometry (GC-MS) was used to quantitatively analyze 22 ingredients in HDs. The experimental results demonstrated that analytes with different polarities exhibited sharp and symmetrical peak shapes on the stationary phase, and all 22 analytes achieved baseline separation on the chromatographic column. The 22 ingredients in HDs showed good linear relationships within their respective ranges, with correlation coefficients greater than 0.9985. The average recovery rates at three spiked levels were in the range of 89.2%-103.2%, and RSDs were less than 5%. Compared with traditional methods, the proposed method has higher efficiency and better accuracy, thus verifying the excellent separation performance of the new stationary phase and the effectiveness of the established GC-MS detection method. The findings indicated the applicability of the developed method to the detection and analysis of various compounds in HDs.

Synthesis and Antibacterial Evaluation of Novel Small-Molecule Antibacterials of a Reduced Acridine Structure in S. aureus Strains Including MRSA.

The increasing antibacterial drug resistance remains a threat to global health with increasing mortality and morbidity. There is an urgent need to find novel antibacterials and develop alternative strategies to combat the increasing antibacterial drug resistance. <P> </P> Objective: We aimed to synthesize novel small-molecule antibacterials to evaluate the structuredependent antibacterial compound activities against S. aureus and MRSA. <P> </P> Method: Compounds were synthesized by primary N-alkylation to form alkyl acridinium salts that were further functionalized with substituted phenyl residues and finally purified by column chromatography. The antibacterial growth inhibition activity was determined as MIC value. <P> </P> Results: The substituent effects on the determined antibacterial growth inhibitory properties have been discussed. <P> </P> Conclusion: The best activities have been found for compounds with methoxy functions, exceeding the activities of reported novel antibacterial peptides. The compounds have also shown antibacterial drug-enhancing effects, which have been manifested as a reduction in the MIC values of the used antibiotics.

Dopamine-supported HPLC post-column derivatization to fluorescence: Simultaneous and sensitive detection of eight tea polyphenols

The effective differentiation and detection of multiple tea polyphenols are often challenging due to their subtle structural similarities. Although post-column derivatization HPLC strategies are commonly employed to distinguish multiple targets, the short physical distance between chromatographic column and detector limits reaction time, thereby reducing the derivatization efficiency. Dopamine (DA) reacts rapidly with resorcinol to form fluorescent azamonardine products, making fast fluorometric derivatization of tea polyphenols containing resorcinol motifs possible. In this study, a DA-driven rapid and post-column fluorescence derivatization method has been applied to sensitively detect eight tea polyphenols. This method is based on fluorescence derivatization and possesses low background interference, high sensitivity, and excellent reproducibility. Moreover, the practical application of this proposed fluorometric derivatization platform was further validated by simultaneous identification of multiple tea polyphenols in different tea samples. This work has great potential to become an alternative to the National Standard method for tea polyphenols determination.

Establishment and evaluation of haloalkane dehalogenase tagged α1A-adrenergic receptor chromatography

Receptor chromatography is an efficient analytical technique that combines the high separation ability of chromatography with the high specificity of receptors for drug recognition. In addition, this technique offers the advantages of active recognition, online separation, and convenient multidimensional target tracking. This strategy allows target active ingredients in complex systems, such as traditional Chinese medicines, to be efficiently screened and accurately identified. Furthermore, the interactions between ligands and immobilized proteins can be studied. To avoid a loss in function, receptor chromatography requires efficient, mild, and simple immobilization methods that do not damage the structure of the immobilized receptors. Improvements in the activity, stability, and ligand-recognition specificity of immobilized functional proteins can be achieved by selecting appropriate immobilization conditions. Notably, the protein immobilization method is not only closely related to the recognition ability of receptor chromatography but also determines the accuracy of the technique. Common methods for immobilizing functional proteins include physical adsorption, chemical reactions, biological affinity reactions, and click chemistry. Despite being easy to operate under mild reaction conditions, these methods have shortcomings, including poor reaction specificity and the necessity of using high-purity functional proteins to prepare chromatography columns. Maintaining the high activity of immobilized receptors and ensuring excellent identification and separation abilities are key challenges in the further development of receptor chromatography. In this work, these issues were addressed by introducing a specific bioorthogonal reaction involving haloalkane dehalogenase (Halo) and 6-chlorohexanoic acid for the immobilization of the α1A-adrenergic receptor (α1A-AR). Specifically, Halo-α1A-AR was immobilized on the surface of 6-chlorohexanoic acid-modified aminopropyl silica gel in one step. The stationary phase with immobilized Halo-α1A-AR was characterized using scanning electron microscopy. Moreover, the activity of the Halo-α1A-AR chromatographic column was evaluated using specific ligands (terazosin hydrochloride, phentolamine mesylate, tamsulosin hydrochloride, and urapidil) and nonspecific ligands (yohimbe and metoprolol) for α1A-AR. Halo-α1A-AR was successfully immobilized on the silica gel surface with good stability over 30 days, and the Halo-α1A-AR chromatographic column exhibited good ligand-recognition activity. The nonlinear chromatography results indicated that prazosin hydrochloride, terazosin hydrochloride, and urapidil interacted with immobilized Halo-α1A-AR through one type of binding site, with association constants of 3.85×105, 5.00×105, and 5.90×105L/mol, respectively. In contrast, phentolamine mesylate and tamsulosin hydrochloride interacted with immobilized Halo-α1A-AR through two types of binding site. The association constants with the high- and low-affinity binding sites were 3.12×106 and 6.01×105L/mol, respectively, for phentolamine mesylate and 9.98×105 and 0.21×105L/mol, respectively, for tamsulosin hydrochloride. Compared with the traditional carbonyldiimidazole method, the immobilization method developed in this work did not require receptor purification and thus minimized the loss of receptor activity. The affinity constants obtained with immobilized Halo-α1A-AR were consistent with the values determined for receptor-ligand binding in solution, indicating that the Halo-α1A-AR chromatography column is suitable for studying drug-protein interactions. This approach also provides a foundation for the efficient screening and accurate determination of target active ingredients in complex systems.