Diverse Effects of Different "Protein-Based" Vehicles on the Stability and Bioavailability of Curcumin: Spectroscopic Evaluation of the Antioxidant Activity and Cytotoxicity In Vitro

Abstract

Background: Curcumin is a natural polyphenolic compound with anti-cancer, antiinflammatory, and anti-oxidation properties. Low water solubility and rapid hydrolytic degradation are two challenges limiting use of curcumin.

Objective: In this study, the roles of the native/modified forms of Bovine Serum Albumin (BSA), β-lactoglobulin (β-lg) and casein, as food-grade biopolymers and also protein chemical modification, in stabilizing and on biological activity of curcumin were surveyed.

Methods: In this article, we used various spectroscopic as well as cell culture-based techniques along with calculation of thermodynamic parameters.

Results: Investigation of curcumin stability indicated that curcumin binding to the native BSA and modified β -lg were stronger than those of the modified BSA and native β -lg, respectively and hence, the native BSA and modified β-lg could suppress water-mediated and light-mediated curcumin degradation, significantly. Moreover, in the presence of the native proteins (BSA and casein), curcumin revealed elevated in vitro anti-cancer activity against MCF-7 (human breast carcinoma cell line) and SKNMC (human neuroblastoma cell line). As well, curcumin, in the presence of the unmodified "BSA and β-lg", was more potent to decrease ROS generation by hydrogen peroxide (H2O2) whereas it led to an inverse outcome in the presence of native casein. Overall, in the presence of the protein-bound curcumin, increased anti-cancer activity and decreased ROS generation by H2O2 in vitro were documented.

Conclusion: It appears that "water exclusion" is major determinant factor for increased stability/ efficacy of the bound curcumin so that some protein-curcumin systems may provide novel tools to increase both food quality and the bioavailability of curcumin as health promoting agent.

Keywords: Curcumin; antioxidant activity; bovine serum albumin; oxidative stress; stability; water exclusion..

Figure 1

(Top) Curcuma Longa with flower and rhizome plus dried turmeric. Turmeric (Curcuma longa L.), belonging to the family of Zingiberaceae, is a perennial herb native to India where its rhizome is used as a yellow colorant curry spice and traditional medicine. (Bottom) Chemical structure of curcumin. The active principle in turmeric was identified as a group of polyphenolic compounds, namely curcumin (74-78%), demethoxy curcumin (15-18%) and bisdemethoxy curcumin (4-6%) commonly referred to as “curcumin”.

Figure 2

The comparison of curcumin stabilityin solvents of water/ethanol under condition of light and darkness (at 420 nm).

Figure 3

The stability of curcumin was investigated at 4, 25, 37 and 80°C temperatures. The experiments performed in 20 mM sodium phosphate buffer, pH 7.0 for 60 min.

Figure 4

(Left) Fluorescence spectra of curcumin in 20 mM sodium phosphate buffer pH 7.0 in the presence of β-lg. Fluorescence spectra of curcumin in 20 mM sodium phosphate buffer pH 7.0 in the absence and presence of different proteins are also shown in Supplementary Figure S1. The concentrations of proteins and curcumin were 40 and 10 μM, respectively. (Right) curcumin stability, based on its fluorescence, in the presence of different proteins (BSA (●), Casein (▲), β-lg (■), modified BSA (○), modified Casein (Δ) and modified β-lg (⧠)) and in buffer alone (×) at 37°C.

Figure 5

Effect of different proteins; native BSA (●), native casein)▲(, native β-lg (■), modified BSA (○), modified Casein (△) and modified β-lg (□) on the curcumin stability against direct light (A) and dark (B), at 25 °C, buffer alone (pH 7, asterisk).

Figure 6

Curcumin-mediated quenching of the native BSA (A), native casein (B), native β-lg (C), modified BSA (D), modified casein (E), and modified β-lg (F) fluorescence at 310 K in 20 mM sodium phosphate buffer pH 7.0. Each data point was the mean of three independent determinations.

Figure 7

Double-logarithmic plot of the curcumin quenching effect on the native BSA (●), native casein (▲), native β-lg (■) (A), modified BSA (○), modified Casein (∆) and modified β-lg (□) (B) fluorescence at 310 K in 20 mM sodium phosphate buffer pH 7.0. K_b and n values are obtained from the y-intercepts and slopes of the plots, respectively.

Figure 8

The antioxidant activity of curcumin alone (⋇) and curcumin in the presence of native BSA (●), native casein)▲(, native β-lg (■), modified BSA (○), modified Casein (∆) and modified β-lg (□) was obtained by measuring of DPPH absorbance at 515 nm, at 4 °C, For 60 h. The concentrations of proteins and curcumin were 6 and 1 μM, respectively, and the concentration was DPPH 0.1 mM. (⧫); curcumin in the presence of Ascorbic acid at equal concentration.

Figure 9

Negative correlation between K_b and stability of curcumin in the presence of native and unmodified proteins in light (A) and dark (B). Also Negative correlation between K_b and anti-oxidant acidity of curcuminin the presence of native and unmodified proteins (C) at 37 °C, buffer alone (pH 7, asterisk).

Figure 10

Cell viability of SKNMC (A-C) and MCF-7 (D-F) cells after exposure to different concentrations of curcumin alone and curcumin-native proteins complexes. Cell viability was determined by MTT assay. Data are expressed as the mean ± S.E.M of three separate experiments (n = 3).

Figure 11

Cell viability of PC12 cells after exposure to different concentrations of free curcumin and curcumin-native proteins complexes (A-C) and curcumin-modified proteins complexes (D-F). Cell viability was determined using MTT assay. Data are expressed as the mean ± S.E.M of three separate experiments (n = 3).

Figure 12

The effect of curcumin on H₂O₂-induced ROS generation. Column bar graph of mean cell florescent for DCF-DA. Data are expressed as the mean ± S.E.M of three separate experiments (n = 3). Control is cells that adding DMSO with them and curcmin control is cells without H₂O₂ and protein and H₂O₂ control is cells without curcumin and protein.