Protection against oxidative stress by rosmarinic acid and its major metabolites in hepatic cells

Background and aims Accumulation of reactive oxygen species causing cellular oxidative stress contributes strongly towards the induction and progression of liver and neurodegenerative diseases [1]. Hence therapeutic focus of recent researches has shifted to antioxidants, with keen interest on those of plant origin. Ocimum canum Sims, an aromatic Ghanaian kitchen spice, is a traditional antidiabetic [2]. Rosmarinic acid (RA), a key phenolic compound of this herb could account for this therapeutic effect [2]. Rosmarinic acid impeded oxidant-induced cellular damage in mice [1]. Hence, antioxidant potential of RA and its metabolites were assayed.

Materials and methods Antioxidant effects of rosmarinic acid (RA) and its key metabolites – caffeic acid (CA), ferulic acid (FA), m-coumaric acid (CoA), 3,4-dihydroxyphenyllactic acid (DPLA) were evaluated in DPPH-radical scavenging assay. Hepatoprotection against oxidant damage induced by 0.5mM tert-butyl hydroperoxide was assayed in HepG2 cells (20 hours pre-exposure and 5 hours co-exposure with chemicals). Quercetin (Q) and DMSO were positive and vehicle controls respectively.

Results

Tab. 1: Comparing the antioxidant potential of phytochemicals in DPPH scavenging assay and cytoprotection assays.

Phytochemical	EC50 (µg/ml) ± SEM
	DPPH	5hr co-exposure	20hr pre-exposure
Quercetin	62.2 ± 2.0	9.2 ± 1.0	29.0 ± 3.10
Rosmarinic acid	80.1 ± 7.6	249.2 ± 12.0α	285.0 ± 62.0α
Caffeic acid	43.4 ± 0.9*#	103.1 ± 10.7αβ	60.1 ± 46.6αβ
3,4-(dihydroxyphenyl)lactic acid	43.6 ± 0.7*#	No effect (> 400 µg/ml)	No effect (> 400 µg/ml)
Ferulic acid	94.7 ± 8.4*#∞	No effect (> 400 µg/ml)	No effect (> 400 µg/ml)
m-Coumaric acid	No effect (> 320 µg/ml)	No effect (> 400 µg/ml)	No effect (> 400 µg/ml)
Antioxidant potencies of phytochemicals in chemical and cellular assays. Values are mean EC50± SEM, n = at least 4 independent assays. *P (< 0.05) when compared to positive control. #P (< 0.0001) compared with rosmarinic acid, ∞P < 0.001 compared with caffeic acid, αP < 0.0001 compared with quercetin and βP < 0.0001 when compared with rosmarinic acid.

Conclusions: CA and DPLA are stronger DPPH-scavengers than the other chemicals; except CoA, having no antiradical activity. With no toxicities, Q protected more effectively than CA and RA in 5hr and 20hr pre-exposure protocols, in decreasing order. FA, CoA and DPLA were ineffective. Despite common theoretical basis, disparity between DPPH and 5hr co-exposure data indicates limitations in cytoprotection. Hence CA is a stronger antioxidant than RA, the parent compound.

References:

[1] Lima, C.F., et al., Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage. Chemico-Biological Interactions, 2007. 167(2): p. 107 – 115.

[2] Berhow, M.A., A.O. Affum, and B.A. Gyan, Rosmarinic Acid Content in Antidiabetic Aqueous Extract of Ocimum canum Sims Grown in Ghana. Journal of Medicinal Food, 2012. 15(7): p. 611 – 620.