The caffeic acid moiety plays an essential role in attenuating lipid accumulation by chlorogenic acid and its analogues

Abstract

Chlorogenic acid (5-caffeoylquinic, CA) possesses distinct hypolipidemic properties in vivo and in vitro, yet the structure-activity relationship (SAR) of CA on lipid metabolism remains unknown. To achieve this aim, we designed and synthesized two sets of CA analogues and evaluated their efficacies to prevent oleic acid (OA)-elicited lipid accumulation in HepG2 cells. Blockage of all hydroxyl and carboxyl groups on the quinic acid moiety did not deteriorate the hypolipidemic effect of CA while blockage of all phenolic hydroxyl groups on the caffeic acid moiety abolished the activity of CA. Further replacement of the quinic acid moiety with cyclohexane and modification of individual phenolic hydroxyl groups on the caffeic acid moiety showed that the phenolic-hydroxyl-reserved analogues displayed a more potent hypolipidemic effect than CA, whereas the analogue with no phenolic hydroxyl displayed little effect on the OA-elicited lipid accumulation. In accordance, the modulating effects of CA on the transcription of the lipogenic gene sterol-regulatory element binding protein (SREBP)1c/1a, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and peroxisome proliferator-activated receptor α (PPARα) were also abolished when the phenolic hydroxyl groups on the caffeic acid moiety were blocked. Our results suggest that the phenolic hydroxyl on the caffeic acid moiety is vital for the lipid-lowering activity of CA.

Fig. 1. The structures of chlorogenic acid, caffeic acid and quinic acid.

Fig. 2. The design of structure–activity relationship research.

Fig. 3. The synthesis of chlorogenic acid analogues. Reagents and conditions: (a) DMP, TsOH; (b) TMSOTf, acetone; (c) BnBr, K₂CO₃.

Fig. 4. The synthesis of a series of cinnamic acid ester derivatives. Reagents and conditions: (e) SOCl₂, cyclohexanol, reflux.

Fig. 5. Effects of CA analogues 4–6 (10 μmol L⁻¹) on OA-elicited lipid accumulation in HepG2 cells. (A) 358 nm measurement after oil-red O staining (n = 8); (B) intracellular TG levels (n = 6); (C) and (D) compounds 4, 5 inhibited OA-elicited lipid accumulation in a dose-dependent way. Data are depicted as means ± SEM. ^###p < 0.001, model vs. blank; *p < 0.05, **p < 0.01, test groups vs. model group. Blk: blank; SimV: simvastatin, used as a positive control; CA: chlorogenic acid; compound 4: 3,4-di-O-isopropylidene-5-O-(3′,4′-dihydroxycinnamoyl)-quinic acid; compound 5: 3,4-di-O-isopropylidene-5-(3′,4′-dihydroxycinnamoyl)-1-O-quinic acid isopropylidene ester; compound 6: 3,4-di-O-isopropylidene-5-O-(3′,4′-dibenzyloxycinnamoyl)-quinic acid benzyl ester.

Fig. 6. Effects of CA analogues 11–14 (10 μmol L⁻¹) on OA-elicited lipid accumulation in HepG2 cells. (A) 358 nm measurement after oil-red O staining (n = 8); (B) intracellular TG levels (n = 6); (C–E) compounds 11, 13, 14 inhibited OA-elicited lipid accumulation in a dose-dependent way. Data are depicted as means ± SEM. ^##p < 0.01, model vs. blank; *p < 0.05, **p < 0.01, ***p < 0.001, test groups vs. model group. Blk: blank; SimV: simvastatin, used as a positive control; CA: chlorogenic acid; compound 11: 3,4-dihydroxycinnamic acid cyclohexanol ester; compound 12: cinnamic acid cyclohexanol ester; compound 13: 3-hydroxycinnamic acid cyclohexanol ester; compound 14: 4-hydroxycinnamic acid cyclohexanol ester.

Fig. 7. Effects of CA and its analogues (10 μmol L⁻¹) on the mRNA levels of lipogenic genes SREBP1c, ACC, FAS, SREBP1a and lipid-oxidation related gene PPARα in OA-elicited HepG2 cells. Real-time quantitative PCR was conducted with gene-specific oligonucleotide primers. The amplification of β-actin served as an internal control (n = 6). Data are depicted as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, test groups vs. negative control group. NC: negative control; CA: chlorogenic acid; compound 5: 3,4-di-O-isopropylidene-5-(3′,4′-dihydroxycinnamoyl)-1-O-quinic acid isopropylidene ester; compound 6: 3,4-di-O-isopropylidene-5-O-(3′,4′-dibenzyloxycinnamoyl)-quinic acid benzyl ester. compound 11: 3,4-dihydroxycinnamic acid cyclohexanol ester; compound 12: cinnamic acid cyclohexanol ester; SREBP1c/1a: sterol response element binding protein 1c/1a; ACC: acetyl-CoA carboxylase; FAS: fatty acid synthase; PPARα: peroxisome proliferator-activated receptor α.