Bioactive Cyclopeptide Alkaloids and Ceanothane Triterpenoids from Ziziphus mauritiana Roots: Antiplasmodial Activity, UHPLC-MS/MS Molecular Networking, ADMET Profiling, and Target Prediction

Abstract

Malaria continues to pose a significant global health burden, driving the search for novel antimalarial agents to address emerging drug resistance. This study evaluated the antiplasmodial potential of Ziziphus mauritiana Lam. (Rhamnaceae) roots through an integrated phytochemical and pharmacological approach. The ethanol extract, along with its derived fractions, demonstrated potent in vitro activity against the chloroquine-sensitive Plasmodium falciparum strain 3D7 (Pf3D7), with the ethyl acetate-soluble (IC₅₀ = 11.35 µg/mL) and alkaloid-rich (IC₅₀ = 4.75 µg/mL) fractions showing particularly strong inhibition. UHPLC-DAD-ESI-QTOF-MS/MS-based molecular networking enabled the identification of thirty-two secondary metabolites (1-32), comprising twenty-five cyclopeptide alkaloids (CPAs), five of which had not yet been described (11, 20, 22, 23, 25), and seven known triterpenoids. Bioactivity-guided isolation yielded thirteen purified compounds (5, 6, 14, 26-30, 32-36), with betulinic acid (30; IC₅₀ = 19.0 µM) and zizyberenalic acid (32; IC₅₀ = 20.45 µM) exhibiting the most potent antiplasmodial effects. Computational ADMET analysis identified mauritine F (4), hemisine A (10), and nummularine R (21) as particularly promising lead compounds, demonstrating favourable pharmacokinetic properties, low toxicity profiles, and predicted activity against both family A G protein-coupled receptors and evolutionarily distinct Plasmodium protein kinases. Quantitative analysis revealed exceptionally high concentrations of key bioactive constituents, notably zizyberenalic acid (24.3 mg/g) in the root extracts. These findings provide robust scientific validation for the traditional use of Z. mauritiana in malaria treatment while identifying specific cyclopeptide alkaloids and triterpenoids as valuable scaffolds for antimalarial drug development. The study highlights the effectiveness of combining advanced metabolomics, bioassay-guided fractionation, and computational pharmacology in natural product-based drug discovery against resistant malaria strains.

Keywords: ADMET profiling; Ziziphus mauritiana; antiplasmodial activity; ceanothane triterpenoids; cyclopeptide alkaloids; in silico pharmacology.

Figure 1

UHPLC-DAD-ESI-MS of the alkaloid-rich fraction of Z. mauritiana.

Figure 2

UHPLC-DAD-ESI-MS of the EtOAc soluble fraction of Z. mauritiana.

Figure 3

SNAP MS molecular networking from the alkaloid-rich fraction. (A): CPA derivatives, (B): indoloquinoline alkaloid derivatives, (C): benzo[a]heptalene alkaloid derivatives, (D): indole alkaloid derivatives, (E): steroid derivatives, (F): steroid peptide derivatives, (G): derivatives of lycotonine-type diterpenoid alkaloids, (H): piperidine and piperazine derivatives, (I): derivatives of cyclic pentapeptide alkaloids, (J): diterpenoids, (K): triterpenoids.

Figure 4

SNAP-MS Subnetwork A from the alkaloid-rich fraction.

Figure 5

SNAP-MS Subnetwork A from the alkaloid-rich fraction after filtering.

Figure 6

Feature-based molecular networking of the alkaloid-rich fraction annotated with GNPS2 ChemWalker tools.

Figure 7

Cyclopeptide alkaloids units.

Figure 8

Cyclopeptide alkaloids of type I core.

Figure 9

(+) ESI MS/MS spectrum of 11.

Figure 10

(+) ESI MS/MS spectrum of 20.

Figure 11

(+) HRESIMS/MS of compound 22.

Figure 12

(+)-ESI MS/MS spectrum of 23.

Figure 13

(+)-ESI MS/MS spectrum of 25.

Figure 14

Chemical structures of putative/identified and isolated compounds (1–36) from Ziziphus mauritiana roots.

Figure 15

Radar chart bioavailability.Lipophilicity: XLOGP3 between −0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Ų, solubility: logS not higher than −6, saturation: fraction of carbons in the sp³-hybridization not less than 0.25, flexibility: no more than nine rotatable bonds.

Figure 16

Pie chart of molecular targets.

Figure 17

XenoSite reactivity model for compounds 4, 10 and 21, including GSH, protein, DNA and cyanide models. Red color indicates the highest scale for probability.

Figure 18

Calibration curve of zizyberenalic acid (32).