Therapeutic effects of self-assembled berberine-glycyrrhizic acid and matrine-glycyrrhizic acid complexes from Qingchang Wenzhong decoction on DSS-induced ulcerative colitis: Mechanisms of anti-inflammatory action.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingchang Wenzhong Decoction (QCWZD), a traditional Chinese medicine (TCM) formula, has been clinically utilized for ulcerative colitis (UC) treatment due to its anti-inflammatory and intestinal barrier-protective effects. However, the mechanisms by which its naturally self-assembled supramolecular complexes, formed during aqueous decoction, contribute to therapeutic outcomes remain underexplored, representing a critical gap in understanding TCM's holistic pharmacology.

AIM OF THE STUDY: This study sought to elucidate the self-assembly mechanisms underlying QCWZD's therapeutic effects, with a focus on characterizing the anti-inflammatory properties of its naturally self-assembled complexes formed during decoction and their role in UC intervention.

METHODS: The submicron precipitate of QCWZD was isolated via gradient centrifugation and characterized for size and morphology using dynamic light scattering (DLS) and scanning electron microscopy (SEM). A DSS-induced murine UC model was used to evaluate the efficacy of QCWZD and its fractions (supernatant and submicron precipitate). Meanwhile, UPLC-ZenoTOF-MS/MS and triple quadrupole LC-MS/MS were employed to identify and quantify the bioactive components. Representative self-assembled complexes were synthesized under conditions mimicking decoction and characterized using DLS and SEM. Thermodynamic binding behavior was probed using isothermal titration calorimetry (ITC). Non-covalent interaction mechanisms of complex formation were characterized by UV, FT-IR, and 1H NMR spectroscopy. Anti-inflammatory activity of the self-assembled complexes was validated in LPS-stimulated RAW264.7 macrophages.

RESULTS: QCWZD's submicron precipitate accelerated UC remission in mice, reducing the disease activity index (DAI), restoring colon length, and suppressing NF-κB p65 phosphorylation compared to the model group. Mass spectrometry identified berberine-glycyrrhizic acid (BER-GL) and matrine-glycyrrhizic acid (MAT-GL) as dominant self-assembled complexes. ITC revealed binding affinity between glycyrrhizic acid (GL) and alkaloids (BER: Kd = 2.55 × 10-4 M, 1:1 binding ratio; MAT: Kd = 6.77 × 10-4 M, 1:2 binding ratio), driven by enthalpy. Charge-transfer and hydrogen-bonding interactions between glycyrrhizic acid (GL) and the alkaloids berberine (BER) and matrine (MAT) lead to stable self-assemblies. BER-GL and MAT-GL complexes significantly suppressed pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) while upregulating IL-10 in RAW264.7 cells, outperforming free alkaloids.

CONCLUSION: Building on QCWZD's established efficacy in treating ulcerative colitis (UC) via the NF-κB pathway, this work demonstrates that the aqueous self-assembly of BER-GL and MAT-GL, derived from QCWZD's submicron precipitate fraction, critically enhances anti-inflammatory efficacy. These findings bridge TCM's traditional preparation methods with supramolecular pharmacology, providing a mechanistic framework for the holistic effects of herbal decoctions. The study advances ethnopharmacological research by highlighting natural self-assembly as a key determinant of TCM's therapeutic synergy.