Rosmarinus officinalis Ethanolic Extracts Rescues BV-2 Cells via Modulating Inflammation and Redox Balance: Comparative Study With Carnosol and Carnosic Acid

Abstract

Neuroinflammation generally refers to an inflammatory response within the central nervous system caused by various pathological insults, including infection, trauma, ischemia, and toxins. As the brain's sentinel immune cell, microglia are tasked as the first responders to infection or tissue injury and initiating an inflammatory response. The perennial shrub plant Rosmarinus officinalis L. was reported to possess anti-inflammatory, anticancer, anti-nociceptive, antidiabetic, neuroprotective, and antioxidative properties. The present study aimed to investigate the effects of Rosmarinus officinalis ethanolic extracts on the lipopolysaccharide (LPS)-induced neuroinflammation model of BV-2 cells in comparison to carnosol and carnosic acid, phenolic diterpenes of the plant. Ultrasound-assisted extraction was used to have ethanolic extract of the plant. LPS was used to induce inflammation in BV-2 cells. Tumor necrosis alpha (TNF-α), interleukin 1 beta (IL-1β) secretion, reactive oxygen species (ROS) production, GSH/GSSG ratio, protein carbonyl level, and caspase-3 activity were evaluated. Inflammation induced by LPS was reduced by the ethanolic extract. Both carnosol and carnosic acid decreased the TNF-α and IL-1β levels as well. The ethanolic extract reduced ROS production and protein carbonylation, and increased GSH/GSSG ratio more effectively compared to the effects of carnosol and carnosic acid. Results depicted that caspase-3 activity was reduced by the ethanolic extract and this effect was more pronounced compared to carnosol and carnosic acid. The present study indicates the ethanolic extract of Rosmarinus officinalis rescues BV-2 cells from apoptosis via alleviating inflammation and oxidative stress.

Keywords: Rosmarinus officinalis L.; apoptosis; inflammation; lipopolysaccharide; microglia; redox modulation.

Figure 1

Chromatograms of Rosmarinus officinalis ethanolic extract. The chemical characterization of the extract was performed with high performance liquid chromatography (HPLC) based on the method of Choi et al. with some modifications [23]. Rosmarinic acid (A), carnosol (B), and carnosic acid (C).

Figure 2

The effect of increased concentrations of Rosmarinus officinalis ethanolic extract. BV‐2 cells were treated with either ethanolic extract (A) or carnosol (B) or carnosic acid (C) for 6, 12, or 24 h and the cell viability was detected with MTT assay. All data (n = 6) were expressed as mean ± SEM; p < 0.05 vs. control (6 h).

Figure 3

The effect of increased concentrations of lipopolysaccharide (LPS) in BV‐2 cells for 6, 12, and 24 h (A). The effect of Rosmarinus officinalis ethanolic extract (Ext), carnosol (CAR), and carnosic acid (CA) in the presence of LPS at 6 and 12 h (B) on BV‐2 cells. MTT assay was performed to investigate the cell viability exposed to the above‐mentioned compounds. All data (n = 6) were expressed as the mean ± SEM; *p < 0.05 vs. control (6 h).

Figure 4

The effect of Rosmarinus officinalis ethanolic extract, carnosol and carnosic acid on oxidative stress in the presence of LPS. ROS levels were detected in BV‐2 cells by measuring the fluorescence of dichlorofluorescein (DCF) (A). Protein carbonyl levels were determined with a commercially available assay kit (B). GSH to GSSG ratio were measured with GSH/GSSG assay kit (C) after 12 h of administration. CA, carnosic acid; CAR, carnosol; Ext, Rosmarinus officinalis ethanolic extract; LPS, lipopolysaccharide. All data (n = 6) are expressed as the mean ± SEM; a: p < 0.05 vs. control, b: p < 0.05 vs. LPS and c: p < 0.05 vs. Ext (7.5 µg/mL).

Figure 5

Evaluation of TNF‐α (A) and IL‐1β (B) levels in BV‐2 cells treated with Rosmarinus officinalis ethanolic extract, carnosol and carnosic acid after 12 h of administration in the presence of LPS. Both markers were detected with ELISA assay using commercially available kits. CA, carnosic acid; CAR, carnosol; Ext, Rosmarinus officinalis ethanolic extract. All data (n = 6) are expressed as the mean ± SEM; a: p < 0.05 vs. control, b: p < 0.05 versus LPS and c: p < 0.05 vs. Ext (7.5 µg/mL).

Figure 6

Detection of caspase‐3 activity in BV‐2 cells treated with Rosmarinus officinalis ethanolic extract, carnosol, and carnosic acid after 12 h of incubation. The activity of caspase‐3 was determined using a commercially available colorimetric assay kit. CA, carnosic acid; CAR, carnosol; Ext, Rosmarinus officinalis ethanolic extract; LPS, lipopolysaccharide. All data (n = 6) are expressed as the mean ± SEM; a: p < 0.05 vs control, b: p < 0.05 vs. LPS and d: p < 0.05 vs. Ext (10 µg/mL).