Indoxyl sulfate (indican) is a tryptophan-derived metabolite generated by intestinal bacteria and subsequently excreted in urine. Urinary indican is considered an indicator of gut microbial imbalance and small-intestinal dysfunction because its production closely reflects the extent of microbial activity. In this study, we improved the classical indican color reaction—originally based on the Obermayer and Jaffé methods—by developing a simple colorimetric method using sodium hypochlorite pentahydrate (NaOCl·5H2O) as a novel oxidizing agent that is characterized by its high stability, strong reactivity, and ease of handling. By using this new oxidizing agent, the overall detection procedure becomes simpler to perform than the classical methods. The addition of hydrochloric acid followed by NaOCl·5H2O produced a blue pigment with an absorbance peak at approximately 600 nm, which was identified as indigo by electrospray ionization mass spectrometry. The method exhibited a visual detection limit of 6 mg/dL, comparable to that of the Obermayer and Jaffé methods. In the proposed method, a linear response between 6 and 12 mg/dL (R2 = 0.9369) was obtained, and the recovery in spiked human urine samples was 69.3%; however, these results indicate that further improvement is required for reliable quantification of elevated indican levels. Moreover, the chromogenic reaction was susceptible to interference from ascorbic acid although partial suppression was achieved using ascorbate oxidase. Overall, this work provides a useful analytical approach for detecting elevated urinary indican levels in clinical and research settings, serving as an alternative to classical methods that rely on ferric chloride and calcium hypochlorite.
Objective Regorafenib (Reg) is a multikinase inhibitor for treating metastatic colorectal cancer (CRC); however, plasma concentrations may not fully predict clinical response. This study aimed to develop and validate an LC‐MS/MS method for quantifying Reg in CRC cells and to preliminarily characterize its temporal and spatial distribution. Methods Separation of Reg and the internal standard (selinexor) was achieved using an Agilent Poroshell 120 EC‐C18 column (3.0 × 100 mm, 2.7 μm) with isocratic elution using acetonitrile and 0.1% aqueous formic acid (70:30, v/v). Detection was performed in multiple reaction monitoring mode using an Agilent 1260‐6460 UHPLC‐MS/MS system. HCT116 and SW480 cells were treated with Reg at their respective IC50 concentrations for 12, 24, 36, and 48 h, and Reg exposure in whole‐cell, nuclear, and mitochondrial fractions was measured. Results The UHPLC‐MS/MS method showed good linearity ( R 2 > 0.99) and a lower limit of quantification of 1.0 ng/mL. Precision (intraday RSD% < 11.39%; interday RSD% < 9.11%) and accuracy (RE% within ±6.80%) were within the acceptance criteria. The recovery ranged from 86.03% to 106.41%, and consistent matrix effects were observed. Reg exposure in whole‐cell samples increased in a time‐dependent manner, while nuclear and mitochondrial fraction‐associated exposures showed distinct temporal patterns between the two CRC cell lines. Conclusion The validated UHPLC‐MS/MS method enables sensitive and reliable quantification of Reg in CRC cells and isolated subcellular fractions. The observed temporal differences in intracellular and fraction‐associated Reg exposure may be associated with cellular response and may support future exposure–response assessments beyond plasma‐based pharmacokinetics.
Reynoutria multiflora (Thunb.) Moldenke is a commonly used herbal drug in traditional Chinese medicine. Prior literature has indicated that R. multiflora possesses broadband pharmacological activities. According to Taiwan Herbal Pharmacopeia (IV) and previous reports, the dominant components of R. multiflora were 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside (THSG) (1) and free anthraquinones such as emodin (2), physcion (3), and rhein (4), which played pivotal roles in the pharmacological effects of herbs. However, the standard analytic method with HPLC-UV took much time to prepare samples and calibration curves based on the standard substances. In this study, a rapid quantitative 1H-NMR analytic method has been developed, which can quantify the indicator compounds in R. multiflora samples. The method validation data demonstrated the good accuracy and precision of this qNMR method. The feasible analytical method can monitor the contents of indicator compounds in both raw and processed herbs. In addition, this method could be applied to analyze the indicator compounds in the decoction Qi Bao Mei Ran Dan successfully. These results all supported the proposed method as a useful tool for quality control of R. multiflora herbal materials and decoctions. With further refinement, this method could serve as a reference for future standard methods in pharmacopoeias.
Equisetum arvense is known as a rich source of phytochemicals, including flavonoids and phenolic acids, in traditional and modern medicine. This study aimed to compare morphological characteristics, phytochemical composition, and antioxidant activity of wild versus aquaponically cultivated E. arvense to assess the impact of cultivation method on plant quality. Plant samples were collected from wild areas and an aquaponic cultivation system. Morphological characteristics were measured. Active compounds were analyzed using HPLC, as well as Folin-Ciocalteu and aluminum chloride methods to measure the concentrations of phenolic compounds and flavonoids. The antioxidant activity of the extracts was also evaluated using DPPH tests. Data were analyzed using an independent t-test. Morphological comparison showed that aquaponic plants had significantly greater height, stem diameter, node spacing, and internode spacing, while wild plants had more leaves per node (p < 0.05 to p < 0.01). No significant differences were observed for internodes per leaf and sheath teeth(ns). HPLC analysis revealed that wild extracts contained higher concentrations of chlorogenic acid (58 mg/g), ferulic acid (11.83 mg/g), and rutin (10.98 mg/g), while aquaponic samples were richer in rosmarinic acid (7.34 mg/g). Total phenolic and flavonoid contents were higher in wild plants (55.4 mg GAE/g and 31.25 mg RE/g) compared to aquaponic ones (45.4 mg GAE/g and 21.25 mg RE/g). Antioxidant activity (IC50) was 282.83 mu g/mL for wild and 182.83 mu g/mL for aquaponic samples. These results suggest that cultivation method critically determines the chemical composition and biological activity of E. arvense. Despite lower phenolic levels, aquaponic plants showed superior antioxidant activity. Each method offers distinct advantages, and neither is absolutely superior. Aquaponic systems may serve as a sustainable platform for producing medicinal plant extracts with potential therapeutic value.
Background Fennel (Foeniculum vulgare Mill.) is widely used as a functional food and plays an important role in traditional medicine worldwide. However, a systematic analysis of its chemical components remains limited.Objective This study aimed to comprehensively identify and analyze the chemical composition of both polar and nonpolar fractions of fennel, specifically its aqueous extracts and volatile oils.Methods Polar metabolites, including phenolic acids, flavonoids, and coumarins, were profiled using ultra-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive-plus-Orbitrap-MS), while gas chromatography-tandem mass spectrometry (GC-MS/MS) was employed for the analysis of volatile constituents.Results A total of 87 compounds were identified in the fennel aqueous extract, including 10 flavonoids, 11 phenolic acids, 3 coumarins, 1 stilbene, 14 alkaloids, 13 organic acids, 7 fatty acids, 10 amino acids, 6 nucleosides, 2 glycosides, 3 terpenoids, and 6 other components (containing 1 sugar alcohol). In addition, 13 compounds were identified in the fennel volatile oil using n-hexane and ethyl acetate as solvents, comprising 11 monoterpenes and 2 phenylpropanoids.Conclusion These findings expand our understanding of the chemical composition of fennel and provide a foundation for elucidating its pharmacodynamic properties.
