Antioxidant, Anti-Inflammatory, Antimicrobial, and Anticancer Activities of Pomegranate Juice Concentrate

Abstract

Pomegranate juice concentrate (PJC) is a rich source of polyphenols, which exhibit significant antioxidant activity and potential health benefits for disease prevention and therapy. In this study, the polyphenolic profile of PJC was investigated for the first time, and it was found that PJC can inhibit oxidative damage to bovine serum albumin (BSA) and deoxyribonucleic acid (DNA), as well as acetylcholinesterase, α-amylase, and tyrosinase activities. The primary polyphenols identified in PJC were 4-Hydroxy-3-Methoxybenzoate, epicatechin, catechin, rutin, ferulic acid, P-coumaric acid, and cinnamic acid. Additionally, PJC demonstrated potent antibacterial effects against human pathogens such as Streptococcus mutans and Aeromonas hydrophila and dose-dependently reduced the proliferation of colorectal, breast, and hepatic cancer cells via apoptosis. Furthermore, PJC blocked B-cell lymphoma 2 (BCl-2) and the expression of a potent cyclin-dependent kinase inhibitor (P21) and enhanced tumor protein (P53) expression, compared to both untreated cells and cells treated with fluoropyrimidine 5-fluorouracil (5-FU). As a result, PJC may be a beneficial ingredient in the formulation of emerging natural-compound-based chemotherapy and functional foods and could be utilized by the food, nutraceutical, and pharmaceutical industries.

Keywords: DNA and protein damage; antibacterial; anticancer; enzyme inhibition; functional food; nutraceutical; pomegranate juice concentrate.

Figure 1

(a) Activity of DPPH^•, ABTS^•, and NO; (b) FRAP assays for PJC, vitamin C, and rutin. Data are expressed as the mean ± standard deviation; n = 3.

Figure 2

Total antioxidant capacity. Assays for PJC, vitamin C, and rutin. Data are expressed as the mean ± standard deviation; n = 3.

Figure 3

Densitometric analysis, 3Dgel, and normal gel for VC, rutin, and PJC. C: Plasmid; Ct.: plasmid + H₂O₂ + UV, plasmid +VC, rutin, and PJC + H₂O₂ + UV; OC: open circular; SC: supercoiled. Data are expressed as the mean ± standard deviation; n = 3.

Figure 4

Densitometric analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a protective effect against the oxidative damage of BSA for VC, rutin, and PJC. C: BSA; Ct: BSA + AAPH, BSA + AAPH + VC, rutin, or PJC. Data are expressed as the mean ± standard deviation; n = 3. Different letters in a column denote significant differences, p < 0.05.

Figure 5

(a) Porcine α-amylase inhibition activity; (b) Tyrosinase inhibition activity of PJC, VC., rutin, acarbose, and kojic acid. Data are expressed as the mean ± standard deviation; n = 3.

Figure 6

Acetylcholinesterase inhibition activity of PJC, VC, rutin, and galantamine. Data are expressed as the mean ± standard deviation; n = 3.

Figure 7

Agar-well diffusion method for antimicrobial activity of PJC at four concentrations against nine different pathogens: 1 (175 µg), 2 (350 µg), 3 (525 µg), and 4 (700 µg). Data are expressed as the mean ± standard deviation; n = 3.

Figure 8

Cytotoxicity profile of PJC against both cancer and normal cell lines. Regular (HSF) cells and cancer (Caco-2, HepG-2, and MDA) cells were exposed to PJC at various dilutions (40–240 μg/mL) for 24 h (A) and 48 h (B). The experiment of the cell viability was evaluated relative to untreated cells using the MTT method. (C) Morphological changes in cancer cells as visualized under the inverted phase-contrast microscope after treatment for 48 h with PJC at different ratios of 40.0, 80, and 160 μg/mL as compared to untreated control cells (0.0 μg/mL). Data are expressed as the mean ± standard deviation; n = 3.

Figure 9

Apoptotic effect of PJC against HepG-2 cells. (A) Fluorescence nuclear staining using PI dye. (B) Fluorescence images of nuclear staining using ethidium bromide-acridine orange of HepG-2 cells. (C) Original flow charts of cell cycle analysis of treated-HepG-2 cells. (D) Quantitative distribution of the treated HepG-2 cells in different phases of the cell cycle. Untreated cells were included as a control reference. HepG-2 cells were exposed to PJC at concentrations of 40, 80, and 160 μg/mL for 48 h.

Figure 10

Estimation of changes in expression levels of four key genes, including B-cell lymphoma 2 (Bcl-2), tumor protein (p53), Catenin beta-1 (β-catenin), and Vascular Endothelial Growth Factor (VEGF), relative to untreated control cells via qPCR. Angiogenesis-related genes are estimated in (A) colon carcinoma (Caco-2), (B) hepatoma (HepG-2), and (C) breast carcinoma (MDA) cells exposed to PJC compared with fluoropyrimidine 5-fluorouracil (5 FU) at IC50 values for 48 h. All values are stated as mean ± SEM and stand for the average values from n = 3.

Figure 11

A color diagram was used to evaluate the correlation heatmap among the contents of TP, TF, phenolic compounds, and antioxidant activity in PJC.