Anti-Inflammatory Activity of Haskap Cultivars is Polyphenols-Dependent

Abstract

Haskap (Lonicera caerulea L.) berries have long been used for their health promoting properties against chronic conditions. The current study investigated the effect of Canadian haskap berry extracts on pro-inflammatory cytokines using a human monocytic cell line THP-1 derived macrophages stimulated by lipopolysaccharide. Methanol extracts of haskap from different growing locations in Canada were prepared and characterized for their total phenolic profile using colorimetric assays and liquid chromatography-Mass spectrometry (UPLC-MS/MS). Human THP-1 monocytes were seeded in 24-well plates (5 × 10⁵/well) and treated with phorbol 12-myristate 13-acetate (PMA, 0.1 μg/mL) for 48 h to induce macrophage differentiation. After 48 h, the differentiated macrophages were washed with Hank's buffer and treated with various concentrations of test compounds for 4 h, followed by the lipopolysaccharide (LPS)-stimulation (18 h). Borealis cultivar showed the highest phenolic content, flavonoid content and anthocyanin content (p < 0.05). A negative correlation existed between the polyphenol concentration of the extracts and pro-inflammatory cytokines: Interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), prostaglandin (PGE2), and cyclooxygenase-2 (COX-2) enzyme. Borealis exhibited comparable anti-inflammatory effects to COX inhibitory drug, diclofenac. The results showed that haskap berry polyphenols has the potential to act as an effective inflammation inhibitor.

Keywords: cytokines; functional food; haskap; inflammation; macrophages; polyphenols.

Figure 1

Sugar and organic acid profile of different haskap cultivars. Results represent the mean ± SD (n = 3), Tukey’s test, p < 0.05. BL, Berry Blue; BR, Borealis; TN, Tundra; IG, Indigo Gem; SAS-TN, Saskatoon Tundra; SAS-IG, Saskatoon Indigo Gem. Significance mean values at p value ≤ 0.05: Capital letter grouping (A–D) is for sugars and small letter grouping (a–c) is for organic acids.

Figure 2

Quantification of TNF-α (A), IL-6 (B), COX-2 (C) and PGE₂ (D) in THP-1 differentiated macrophages. The cells were pretreated with test compounds for 4 h, followed by 18 h LPS-stimulation. Results represent the mean ± SD (n = 3), Tukey’s test, p < 0.05. Cont, control; LPS, lipopolysaccharide; BR, borealis; BL, berry blue; IG, indigo gem; TN, tundra; Diclo, diclofenac; Nime, nimesulide. For each bar, different letters a-g represents significance statistical difference using Tukey’s t-test analysis (p ≤ 0.05). The bars with one or more same letter are not statistically different.

Figure 3

Inhibition of free-radical product (nitric oxide) by haskap berry extract in THP-1 differentiated macrophages. The cells were pretreated with test compounds for 4 h, followed by 18 h LPS-stimulation. Results represent the mean ± SD (n = 3), Tukey’s test, p < 0.05. Cont, control; LPS, lipopolysaccharide; BR, borealis; BL, berry blue; IG, indigo gem; TN, tundra; Diclo, diclofenac; Nime, nimesulide. For each bar, different letters a–e represents significance statistical difference using Tukey’s t-test analysis (p ≤ 0.05).

Figure A1

Morphology of human monocytes (THP-1) and LPS-induced macrophages. Monocytes exist in suspension phase and have spherical shape. Monocytes upon differentiation take adherent phase and form various cell shapes. The undifferentiated monocytes (A), (B) and differentiated macrophages (C), (D) are shown in the figure.