The study combined high-resolution liquid chromatography with tandem mass spectrometry to profile secondary metabolites in methanolic extracts of L. arborea leaves and stalks and in three leaf-derived fractions. LC-ESI/LTQ-Orbitrap-MS/MS permitted the tentative identification of 14 compounds, including flavonoids, a phenolic derivative, fatty acids, oxygenated fatty acids and a diterpene glycoside. The researchers then isolated five major flavonoids and unequivocally characterized them by one- and two-dimensional nuclear magnetic resonance spectroscopy, confirming annotations initially based on accurate mass, retention time and fragmentation patterns.

Plant material was collected in Aswan Governorate, Egypt, in November 2023 during flowering and fruiting, and a voucher specimen was deposited at Aswan University. Leaves and stalks were separated, air-dried at 36 °C, ground and macerated separately with 80% methanol for 48 hours, with two extractions performed for each material. The crude leaf extract was subsequently partitioned on a reversed-phase C18 column with a stepwise methanol gradient. The resulting 40%, 60% and 80% methanol fractions were designated LaLA, LaLB and LaLC, while the original leaf and stalk extracts were designated LaL and LaS. Preliminary thin-layer chromatography detected flavonoid derivatives and glycosylated compounds in all three leaf fractions.

For chemical profiling, extracts were analyzed in negative electrospray mode using reversed-phase ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry. The method covered a mass range of 120 to 1,600 m/z, with data-dependent acquisition selecting abundant precursor ions for fragmentation at a normalized collision energy of 30%. Feature detection and alignment used a mass tolerance below 5 parts per million, a signal-to-noise ratio above 3 and a retention-time window of plus or minus 0.2 minutes. Peak areas were normalized to total ion current, and identifications relied on accurate mass, elemental composition, isotope-pattern agreement and comparison of tandem mass spectra with literature on the Apocynaceae family.

Flavonoids predominated in the profile, chiefly as O-glycosides. Diagnostic fragment ions supported tentative assignments of apigenin-6,8-di-C-glycoside, luteolin-7-O-β-glucopyranoside, apigenin-7-O-β-glucopyranoside, luteolin and apigenin, as well as rutin and chrysoeriol. The researchers isolated apigenin-6,8-di-C-glycoside, luteolin-7-O-β-glucopyranoside, apigenin-7-O-β-glucopyranoside, luteolin and apigenin, then used detailed NMR data to determine their structures, including glycosylation positions and sugar identities. This structural confirmation strengthened the LC-MS assignments and reduced the possibility that structurally similar isomeric glycosides had been confused on mass-spectrometric evidence alone.

Beyond flavonoids, the analytical workflow detected coumaroyl-O-glucaric acid, azelaic acid, a compound assigned as steviolbioside, three oxylipins and dihydroxypalmitic acid. The oxylipin assignments were based partly on a diagnostic product ion at m/z 171, while sequential sugar losses supported the steviolbioside annotation. Most compounds appeared across the leaf extract, stalk extract and the three leaf fractions, although one oxylipin was detected only in the 80% leaf fraction and stalk extract, and dihydroxypalmitic acid was absent from the 40% fraction. The authors reported coumaroyl-O-glucaric acid, steviolbioside and the polar lipid derivatives as new descriptions within the genus Leptadenia.

For the biological tests, blood was obtained with consent from healthy, nonsmoking volunteers aged 19 to 24 who reported no alcohol use and no medication, antiplatelet medicine or antioxidant supplementation during the preceding two weeks. Plasma and platelets were incubated for 30 minutes at 37 °C with extracts, fractions or isolated compounds, with extract and fraction concentrations ranging from 0.5 to 50 micrograms per milliliter and pure compounds tested at 5 micrograms per milliliter. Oxidative stress was induced with 4.7 mM H2O2, 3.8 mM Fe2+ and 2.5 mM EDTA. The researchers measured thiobarbituric acid-reactive substances as an indicator of lipid peroxidation, protein carbonyl groups as a marker of protein oxidation and free thiol groups as a measure of thiol oxidation.

The 80% leaf fraction had the broadest statistically significant antioxidant profile at 5 micrograms per milliliter, significantly lowering lipid peroxidation, protein carbonylation and thiol oxidation in both plasma and platelets. The 60% fraction also showed substantial activity against platelet lipid peroxidation and protein carbonylation, and the 40% fraction was effective in several assays, although responses were not uniformly significant. At 50 micrograms per milliliter, all three fractions significantly inhibited platelet lipid peroxidation by about 60%. Extracts and fractions did not appreciably change the measured biomarkers in samples lacking the oxidative-stress inducer, indicating that the observed effects occurred under the imposed stress condition rather than as a general alteration of baseline values.

Five purified flavonoids also showed antioxidant activity in the comparison experiment. At 5 micrograms per milliliter, apigenin, luteolin, apigenin-6,8-di-C-glycoside, apigenin-7-O-glucopyranoside and luteolin-7-O-β-glucopyranoside reduced one or more indicators of oxidative damage in plasma or platelets, with significant effects reported in the relevant models. For example, apigenin reduced plasma lipid peroxidation by about 47% and plasma protein carbonylation by about 34%, while it increased free thiols by about 42% in plasma and 45% in platelets. These purified compounds were often less effective than some plant preparations, which the authors interpreted cautiously as possible evidence of combined or synergistic effects among multiple metabolites rather than proof of a specific mechanism.

Whole-blood testing produced a different pattern. Blood was passed through a collagen-coated PL-chip in the Total Thrombus-formation Analysis System, with secondary hemostasis blocked so that the assay focused on primary hemostasis under shear. Only the 40% leaf fraction significantly increased the 10-minute area under the pressure curve, by approximately 20%, at concentrations of 5 and 50 micrograms per milliliter. This represented greater thrombus formation in that in vitro system. Aspirin, used as a reference, reduced the area under the curve by about 50% and significantly prolonged occlusion time. None of the other extracts or fractions significantly modulated thrombus formation, separating the antioxidant findings in plasma and platelets from the procoagulant response observed for one fraction in whole blood.

The findings provide a detailed chemical and experimental basis for further studying L. arborea, a climbing plant whose leaves, shoots and young fruits are consumed as a nutritious green vegetable in parts of the Sahel and Arabian Peninsula. However, the work remains limited to laboratory assays using human samples from volunteers in a narrow age group, with no in vivo or clinical evaluation reported. The PL-chip does not identify individual platelet activation pathways, while the possible influence of digestion, metabolism and bioavailability on the extracts and isolated phenolics remains unresolved. The authors called for further in vitro and in vivo research to determine whether the measured antioxidant effects and modulation of hemostasis correspond to biologically relevant outcomes.

Subject of Research: In vitro phytochemical, antioxidant and hemostatic assessment of Leptadenia arborea leaf and stalk preparations

Article Title: LC-HRMS/MS-based qualitative secondary metabolites profiling of Leptadenia arborea and evaluation of its antioxidant and hemostatic activities

Article References: Olas, B., Rashidi, N. A., Cerulli, A., Mohamed, A. A. A., Kontek, B., Sławińska, N., Kalamat, K., Abdel-Farid, I. B., Piacente, S., & Hamed, A. I. (2026). LC-HRMS/MS-based qualitative secondary metabolites profiling of Leptadenia arborea and evaluation of its antioxidant and hemostatic activities. Frontiers in Molecular Biosciences, 13, 1951196. https://doi.org/10.3389/fmolb.2026.1951196

Image Credits: AI Generated

DOI: 10.3389/fmolb.2026.1951196

Keywords: Leptadenia arborea, flavonoid glycosides, LC-HRMS/MS, NMR spectroscopy, oxidative stress, human plasma, blood platelets, whole blood, thrombus formation, hemostasis, LC-HRMS, MS-based