Alzheimer's disease (AD) is a devastating neurodegenerative disease caused by multiple factors. Single-target drugs have limited efficacy for AD treatment. Therefore, multi-target intervention strategy has great potential. Traditional Chinese medicine (TCM) is mostly used in the form of compound prescription, which has the pol-ypharmacology behavior. Rhizoma Coptidis and Radix et Rhizoma Rhei are frequently used as the couplet medi-cines of TCM for AD therapy. In this study, the novel carrier-free nanoassembly with 3D-porous frame crystal structure has formulated from supramolecular self-assembly of berberine (BER) and rhein (RHE), the main active components of Rhizoma Coptidis and Radix et Rhizoma Rhei, respectively. Combining with the spectral data and single crystal structure, the self-assembly process was clarified as dominated by electrostatic interaction and 7C-7C stacking. In vitro release property, cholinesterase (ChE) inhibition, ll-amyloid (All) aggregation regulation, radical elimination, metal ions chelation and cytotoxicity assay indicated that the obtained BER-RHE assembly had the Fickian diffusion-controlled sustained release ability, synergistic biological activities and virtually no neuro-toxicity. In addition, in vivo reactive oxygen species (ROS) level evaluation showed that the assembly could reduce the accumulation of intracellular ROS in Caenorhabditis elegans (C. elegans). Meanwhile, BER-RHE as-sembly could also be used as a novel potential carrier for drug delivery due to its superior 3D-porous frame. This green and facile strategy for carrier-free nanoassembly microscopic construction via supramolecular self -assembly might provide inspiration for the development of multi-target therapy for AD and the design of the novel drug delivery system.
