Ziziphusine A, a unique plant-alkaloid featuring amide rotational isomerism from the fruits of Ziziphus jujuba with anti-inflammatory activity.

Ziziphus jujuba var. inermis Rehder (Rhamnaceae) is an edible medicinal plant widely used in traditional medicine and functional foods. Phytochemical investigation of the methanolic extract of its fruits resulted in the isolation of a rare amide rotational isomeric alkaloid, ziziphusine A (1), together with eight known compounds including brassicasterol (2), (E)-24-nor-cholest-22-en-3β-ol (3), alphitolic acid (4), 3-O-cis-p-coumaroyl alphitolic acid (5), 3-O-trans-p-coumaroyl alphitolic acid (6), E-p-methoxycinnamic acid (7), (Z)-3-(4,5-dihydroxy-2-methoxyphenyl)acrylic acid (8), and scopoletin (9). Their structures were elucidated by spectroscopic analyses including 1D and 2D NMR and HR-ESIMS data, in combination with comparison to literature values. Compound 1 represents a unique naturally occurring case of amide bond rotation isolated from plant natural products. All isolated compounds were evaluated for inhibitory effects on nitric oxide production in LPS-stimulated RAW264.7 macrophages and for antimicrobial activity against six bacterial strains. Compound 1 exhibited significant NO inhibitory activity with an IC50 value of 19.1 µM and suppressed the pro-inflammatory mediators iNOS and COX-2, whereas compounds 2 and 3 showed moderate activity with IC50 values of 42.2 and 37.8 µM, respectively. None of the tested compounds displayed significant antimicrobial activity at 25 µM. Molecular docking studies demonstrated favorable interactions of 1 with the inflammatory enzymes iNOS and COX-2, with binding energies ranging from -7.6 to -8.0 kcal mol-1. In addition, molecular dynamics simulations and binding free-energy analyses were employed to further evaluate the binding stability and dynamic conformational behavior of ziziphusine A with iNOS and COX-2 complexes. Furthermore, in silico ADMET and toxicity prediction suggested favourable pharmacokinetic properties with manageable predicted toxicity risks of ziziphusine A. These findings highlight ziziphusine A as a structurally unique natural product with promising anti-inflammatory potential.