Amelioration of atherosclerotic complications and dyslipidemia by verbascoside-enriched fraction of Clerodendrum glandulosum leaves targeting LDL-R and LXR-mediated reverse cholesterol transport.

OBJECTIVE: Clerodendrum glandulosum is widely used in traditional Chinese and Indian systems of medicine for conditions like hypertension and diabetes. While various pharmacological benefits have been reported, research on its anti-atherosclerotic properties remains limited. Atherosclerosis (AS) is a chronic cardiovascular disease linked to dyslipidemia (DLD) and inflammation. This study aims to identify the bioactive fraction from C. glandulosum extract, evaluate its potential against AS and DLD, and explore the molecular mechanisms of cholesterol metabolism. METHODS: Bioactivity-guided fractionation was employed to investigate the bioactivity of C. glandulosum by screening biochemical enzyme inhibitory potential. The active fraction was subjected to in vitro testing to assess the anti-inflammatory and anti-adhesion properties. The fraction was administered at 50 and 100 mg/kg per os (p.o.) to cholesterol-cholic acid-thiouracil (CCT) diet-induced atherosclerotic Wistar rats. Changes in lipid and antioxidant profiles, inflammatory markers, and cholesterol metabolism pathways were assessed using Western blotting. Histopathological analyses of the aorta, liver, heart, and kidneys were also conducted. Molecular docking was conducted for the verbascoside (VER) and the standard statin, atorvastatin (ATS), for their binding capabilities with the molecular targets considered in this study. RESULTS: Bioactivity-guided fractionation and screening revealed that ethyl acetate fraction (EAF) contained VER as the principal phytoconstituent. EAF exhibited potent enzyme inhibitory activity, with IC(50) values of 1.059 mg/mL for pancreatic lipase and 22.48 µg/mL for α-glucosidase. In vitro analysis revealed that EAF significantly lowered cell-to-cell adhesion to 0.57 folds from 2.5 folds in the disease control and normalized the inflammatory cytokines. In CCT-diet-induced rats, elevated serum cholesterol and low-density lipoproteins (LDL) levels (92.1 mg/dL and 78.49 mg/dL, respectively) were reduced to 63.52 mg/dL and 58.51 mg/dL with EAF at 100 mg/kg. EAF at 100 mg/kg reduced oxidized LDL to 53.63 ng/mL compared to 157.1 ng/mL in CCT-diet-fed rats. EAF also restored antioxidant activity by increasing superoxide dismutase and catalase levels to 73.78 and 17.72 U/mg protein, respectively, compared to 42.22 and 9.62 U/mg protein in CCT-diet-fed rats. EAF restored inflammatory cytokines to normal levels. Histological analyses validated the protective benefits of EAF supplementation for the structural integrity of the aorta, liver, heart, and kidney tissues. Western blotting analysis of liver tissues revealed changes in the cholesterol metabolic pathway by upregulating peroxisome proliferator-activated receptor gamma (PPARγ)/liver X receptor alpha (LXRα)/adenosine triphosphate-binding cassette sub-family G member 1 (ABCG1) and low-density lipoprotein receptor (LDL-R) expression. Unlike ATS, molecular docking analyses indicated strong interactions between VER and molecular targets. CONCLUSION: EAF prevented DLD and AS by reverse cholesterol transport via the PPARγ/LXRα/ABCG1 pathway, offering potential therapeutic benefits for cardiovascular health.