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Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06–625.00 ng/mL. The intra- and inter-assay precisions were 0.74–2.98% and 6.57–9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70–110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.

The rising global incidence of obesity underscores the urgent demand for effective therapeutic interventions. Natural products have emerged as promising alternatives; however, identifying candidates that effectively target the complex mechanisms underlying obesity remains a critical challenge. In this study, the specialized metabolites of Dendrobium albosanguineum were investigated for their anti-obesity potential. Methanolic extraction was performed on the entire plant, followed by systematic fractionation and compound elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy. A set of in vitro colorimetric assays was employed to assess pancreatic lipase inhibition, cytotoxicity, intracellular lipid storage, triglyceride content, and glycerol release in murine (3T3-L1) and/or human (PCS-210-010) adipocyte models. In addition, flow cytometry, western blotting analysis, and RT-qPCR were used to evaluate the effects of a chosen metabolite on cell cycle progression and the expression of adipogenesis-related genes and proteins. Eight metabolites were isolated, including bibenzyls (moscatilin, chrysotoxine), lignans (syringaresinol, foliachinenoside C), a sterol (daucosterol), a phenylpropanoid ( n -octacosyl- trans - p -coumarate), and flavonoids (rhoifolin, kaempferol-3- O -(2″,6″-dirhamnosyl)glucoside). Among them, foliachinenoside C exhibited the most potent activity, with 94.77 ± 0.85% inhibition of pancreatic lipase (IC 50  = 40.73 ± 0.74 µM). It significantly reduced triglyceride levels and promoted glycerol release in both murine and human adipocytes. Mechanistically, foliachinenoside C induced cell cycle arrest at the G0 phase in 3T3-L1 cells and downregulated key adipogenic transcription factors (PPARγ, C/EBPα, SREBP1c) and lipogenic proteins (FAS, PLIN1, LPL, ADPN, FABP4). Moreover, it modulated the AKT/GSK3β and AMPK-ACC signaling pathways, collectively suppressing adipocyte differentiation. These findings position foliachinenoside C as a promising plant-derived compound for obesity pharmacotherapy, warranting further investigation to facilitate its clinical development.

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum ) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix -containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95–125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph–mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix .

Background: Honey has been widely used as a traditional vehicle of herbal medicines. Honey behaves as a natural deep eutectic solvent (NADES) containing β-glucosidase; therefore, it can be used for the extraction and bio-activation of the bioactive compounds of herbs. Objectives: This study aims to apply honey (H-NADES) and a sugar-based NADES (S-NADES) for the extraction, analysis, and bioconversion of daidzin from Pueraria candollei var. mirifica (PM) root. Materials and Methods: Various concentrations of H-NADES and S-NADES (water:sucrose:glucose: fructose, 18:3:18:22 by weight) were used as solvents for extraction. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and validated for monitoring the extraction efficacy. The catalytic reactivity against daidzin of β-glucosidase purified from honey was investigated. Results: Using NADESs as solvents, icELISA was suitable for the reliable determination of daidzin with high sensitivity (1.95-125 ng/mL), specificity (% cross-reactivity ≤ 2.60), and accuracy (98.3-106% daidzin recovery). Daidzin at a concentration of 75.8 ± 3.67 μg/mL was extracted using 50% (v/v) S-NADES, which was the most effective for the extraction compared to H-NADES, water and ethanol. In addition, daidzin was converted to daidzein by honey β-glucosidase. Conclusion: Both S-NADES and H-NADES were useful for the extraction, analysis, and bioconversion of daidzin, and β-glucosidase from honey might enhance the oestrogenic activity and bioavailability of PM phytochemicals.

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.