Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells

Abstract

Microglial activation can release free radicals and various pro-inflammatory cytokines, which implicates the progress of a neurodegenerative disease. Therefore suppression of microglial activation can be an appropriate strategy for combating neurodegenerative diseases. Betanin is a red food dye that acts as free radical scavenger and can be a promising candidate for this purpose. In this study, purification of betanin from red beetroots was carried out by normal phase colum chromatography, yielding 500 mg of betanin from 100 g of red beetroot. The purified betanin was evaluated by TLC, UV-visible, HPLC, ESI-MASS, FT-IR spectroscopy. Investigation on the inhibitory effect of betanin on activated microglia was performed using primary microglial culture. The results showed that betanin significantly inhibited lipopolysaccharide induced microglial function including the production of nitric oxide free radicals, reactive oxygen species, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). Moreover, betanin modulated mitochondrial membrane potential, lysosomal membrane permeabilization and adenosine triphosphate. We further investigated the interaction of betanin with TNF-α, IL-6 and Nitric oxide synthase (iNOS or NOS2) using in silico molecular docking analysis. The docking results demonstrated that betanin have significant negative binding energy against active sites of TNF-α, IL-6 and iNOS.

Fig 1. Purification and characterization of betanin.

Evaluation of purified betanin. TLC (i), Optical absorption (ii), and FT-IR (iii). Comparison of purified and standard betanin. HPLC chromatogram (iv), Mass spectrum (v), Antioxidant activity using DPPH assay (vi).

Fig 2. Cell isolation, cell viability and investigation of NO• level.

Flow cytometry analysis of CD-11b+ cells (Fig i). Cytotoxicity of betanin on microglial cells using the MTT assay (Fig ii). The control group is the cells treated with PBS as betanin solvent. Evaluation of LPS-induced microglial cells. Morphology characteristics of microglia cells before and after LPS induction with 200x magnification light microscope (Fig iii). Before induction, cells are healthy and ramified (a), after LPS-induction, cells switch to the inflamed and amoeboid form (b). Determination of nitric oxide by Griess reaction (iv). All groups were compared with the control group. The difference in NO^• concentration in the LPS group and control group was more than five times. However, no significant difference was detected between the control and 500 μM betanin. *P<0.05, **P<0.01, ***P<0.001 and #P>0.05 or non-significant, one-way ANOVA.

Fig 3. ROS, MMP, ATP, and redistribution of lysosomes analysis.

Flow cytometry, ATP, and LMP analysis of microglial cells before, and after pretreatment with betanin at 500 μM concentration. Flow cytometry histogram of ROS (i) and MMP (ii), comparison between control and pretreated (left), control and LPS-induced groups (right). ATP levels (iii). LMP analysis by acridine orange redistribution. The absorbance of the control group was considered as one (iv). *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA.

Fig 4. Evaluation of pro-inflammatory cytokines.

Comparison of cytokines levels in microglial cells after pretreatment with 500 μM of betanin. Concentration of TNF-alpha: 82, 418, 75 pg/ml, IL-6: 261, 705, 363 pg/ml and IL-1BETA: 2573, 3610, 1553 pg/ml were obtained for control, LPS and LPS+Betanin groups, respectively. The experiment was performed with three replications at two different times. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA.

Fig 5. Comparison of Tnf-α, Nos2 and Nf-кb expression.

Changes in gene expression of Tnf-α, Nos2 and Nf-кb in microglial cells under the influence of betanin pretreatment at 500 μM. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA.

Fig 6. Protein modeling and docking study.

Ramachandran and RMSD analysis plots of IL6, iNOS, TNF-α and NF-кB proteins modeling showed that, these structures did not contain any residues in the disallowed regions. According to the RMSD values these proteins were stable in the simulation time period. The visual illustration showed the binding energy between betanin and IL6 (ΔG = -7.3 kcal/mol), iNOS (ΔG = -9.1 kcal/mol), TNF-α (ΔG = -9.6 kcal/mol) and NF-кB (ΔG = -8.5 kcal/mol). The docked pose of betanin and each protein showed the key hydrogen-bonds area by using AutoDock Vina results. All visual illustration of the interactions generated with Discovery Studio visualizer 19.1.0.219 (https://www.3dsbiovia.com/products/collaborative-science/biovia-discovery studio/visualization-download.php) as a free resource.