An exploratory investigation into the physicochemical, antioxidant and cellular effects of a selection of honey samples from the Southern African region

Abstract

The unique floral biodiversity of Southern Africa would be reflected in the phenolic acid and flavonoid composition as well as the antioxidant activity of honeys from this region. In this exploratory investigation the total polyphenolic (TPC) and flavonoid (TFC) content, antioxidant activity as well as the cellular protective effects of a selection of honeys collected in this region was evaluated. Thirteen honey samples representative of the Western Cape (WCa, WCb and WCc), Eastern Cape (ECa, ECb and ECc), South East Mozambique (SEMa, SEMb and SEMc) and Agricultural: A-E (Eucalyptus) (A-E1 and A-E2), A-L (Litchi) and A-O (Orange) were collected. These samples were subjected to physicochemical analysis, the antioxidant content (TPC and TFC) and both enzymatic (catalase activity) and non-enzymatic activity, using the 2,2-diphenyl-2-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays was determined. From the DPPH, TEAC and ORAC data the Relative Antioxidant Capacity Index (RACI) was calculated. To determine whether high antioxidant activity translates into significant cellular protection, biological and cellular assays were undertaken. Using the pBR322 plasmid assay and the erythrocyte haemolysis assay the ability of honeys to protect against 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) oxidative damage was evaluated. Further evaluation was undertaken in the SC-1 fibroblast cell line and the physiologically more relevant Caco-2 cell line. Toxicity and antioxidant effects were evaluated in the SC-1 cell line while antioxidant effects were only evaluated in the Caco-2 cell line. The long-term mitogenic and toxic effects were determined in the SC-1 cell line using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Neutral Red (NR) and Crystal Violet (CV) assays. Short term, total- and intracellular antioxidant effects were determined in both cell lines using the dichlorofluorescein diacetate assay (DCFH-DA) assay. For all cellular experiments honey at concentrations of 0.01% and 1% were used. The physiochemical properties of the honeys evaluated fulfilled the regulatory standards compiled in the Codex Alimentarius (CODEX STAN12-1981 revision 2001). The results were as follows: SEMb had the highest TPC (167.96 mg GAE/100g) and TFC (51.60 mg CE/100g) while A-E2 had the highest catalase (38.48 µmol H2O2/g) activity. RACI revealed that WCb had the highest antioxidant activity.SEMc showed the highest protection of plasmid DNA against oxidative-induced strand breaks while SEMa showed the highest protection of erythrocytes against AAPH-induced haemolysis. Although correlations were found between antioxidant content and antioxidant activity assays, no correlation was found these parameters and the biological assays. For the long-term cytotoxicity assay, AAPH showed significant cytoxicity at 0.78mM, 1.56mM and 0.28mM when measured using the MTT, NR and CV assays, respectively. Some honeys 4/13 and 3/13 showed a mitogenic effect at a concentration of 0.01% and 1% respectively. Toxic effects, were observed for 1/13 and 8/13 at 0.01% and 1% honey respectively. Toxicity after 72 h exposure varied from 10-30% (CV assay). The same concetrations of honey was used to determine the short-term, 2h, antioxidant effects in both the SC-1 and Caco-2 cell lines. No oxidative effect was found for all honeys at these concentrations. For the DCFH-DA assay using the SC-1 cell line at 1%, 12/13 and 7/13 honeys showed total and intracellular protection respectively. The highest extracellular protection was for SEMa (% Protection (%P) = 95) and SEMb (%P = 93). Intracellular protection was the highest for SEMc (%P = 21) and A-L (%P = 20). At 0.01%, 7/13 and 8/13 honeys exhibited total and intracellular protection, respectively. For both the highest protection was found for SEMc (%P = 43, total and %P = 30, intracellular). For the Caco-2 cell line at 1%, 11/13 and 4/13 showed total and intracellular protection, respectively. Of these the highest extracellular protection was for SEMb (% Protection (%P) = 90). Intracellular protection was the highest for ECa (%P = 28) and WCc (%P = 26). At 0.01%, 4/13 and 8/13 honeys showed total and intracellular protection respectively. The highest extracellular protection was found for SEMc (%P = 62) and intracellular protection was ECc (%P = 28). The SC-1 cell line was found to be the most sensitive to the antioxidant effects of honey compared to the Caco-2 cell line. The honeys SEMa, SEMb and SEMc showed protection against oxidative damage in both cell lines. In conclusion, the antioxidant activity of honeys from Southern Africa is of a high quality. The WC, SEM and EC honeys showed the highest antioxidant effects and could provide health benefits against diseases associated with oxidative stress as indicated by these results. Copyright