BIOASSAY-GUIDED FRACTIONATION OF LEPIDIUM SATIVUM L. SEEDS REVEALS A FLAVONOID-AGLYCONE SUB-FRACTION WITH DUAL ANGIOTENSIN-CONVERTING ENZYME INHIBITORY AND ENDOTHELIAL NITRIC OXIDE STIMULATORY ACTIVITY

Abstract

Arterial hypertension affects an estimated 1.28 billion adults worldwide and its cardiovascular sequelae remain a leading driver of premature mortality.[1] Simultaneous pharmacological engagement of the renin–angiotensin–aldosterone system (RAAS) and the endothelial nitric oxide (NO) pathway offers a mechanistic rationale for more complete vascular protection than single-target agents provide. Seeds of Lepidium sativum L. (family Brassicaceae; garden cress) are incorporated into Moroccan, Indian and Arabian traditional medicine for the management of elevated blood pressure, yet no bioassay-guided phytochemical study with simultaneous dual-target in vitro screening has been reported. Successive Soxhlet extraction of authenticated L. sativum seeds (500 g) in four solvents of ascending polarity yielded four chemically distinct extracts: n-hexane (LSH, 8.62% w/w), ethyl acetate (LSEA, 3.41%), ethanol (LSE, 11.85%) and hydroalcoholic 70:30 v/v (LSHA, 14.27%). Each extract was characterised by qualitative phytochemical screening and HPTLC fingerprinting on silica gel 60 F254 plates against seven authenticated reference standards. All four extracts were evaluated for angiotensin-converting enzyme (ACE) inhibitory activity by the hippuryl-L-histidyl-L-leucine (HHL) spectrophotometric assay and for endothelial NO stimulation in human umbilical vein endothelial cells (HUVECs) by the Griess reaction. The ethanol extract (LSE) emerged as the most active in both assays: ACE IC50 89.4 ± 4.2 µg/mL and HUVEC nitrite stimulation of 212.1% over untreated control at 50 µg/mL (approximately 86% of the L-arginine substrate response). Bioassay-guided silica gel column chromatography of LSE yielded three pooled sub-fractions; the flavonoid-aglycone-enriched sub-fraction LSE-F2 — dominated by quercetin (Rf 0.45) and kaempferol (Rf 0.52) by HPTLC achieved an ACE IC50 of 31.5 ± 1.9 µg/mL, representing a 2.84-fold enrichment over the parent extract. Convergent ACE-inhibitory and NO-stimulatory rank orders across all extracts and sub-fractions, together with the compositional–activity correlation established by column fractionation, are consistent with quercetin and kaempferol acting as principal pharmacophores at both vasoactive targets. These findings provide a molecular rationale for the antihypertensive ethnopharmacology of L. sativum seeds and identify LSE-F2 as a candidate fraction warranting in vivo pharmacological validation.