Ocimum basilicum L. Methanol Extract Enhances Mitochondrial Efficiency and Decreases Adipokine Levels in Maturing Adipocytes Which Regulate Macrophage Systemic Inflammation

Abstract

Excessive storage of lipids in visceral or ectopic sites stimulates adipokine production, which attracts macrophages. This process determines the pro- and anti-inflammatory response regulation in adipose tissue during obesity-associated systemic inflammation. The present study aimed to identify the composition of Ocimum basilicum L. (basil) seed extract and to determine its bio-efficacy on adipocyte thermogenesis or fatty acid oxidation and inhibition of lipid accumulation and adipokine secretion. Ocimum basilicum L. seed methanol extract (BSME) was utilized to analyze the cytotoxicity vs. control; lipid accumulation assay (oil red O and Nile red staining), adipogenesis and mitochondrial-thermogenesis-related gene expression vs. vehicle control were analyzed by PCR assay. In addition, vehicle control and BSME-treated adipocytes condition media were collected and treated with lipopolysaccharide (LPS)-induced macrophage to identify the macrophage polarization. The results shown that the active components present in BSME did not produce significant cytotoxicity in preadipocytes or macrophages in the MTT assay. Furthermore, oil red O and Nile red staining assay confirmed that 80 and 160 μg/dL concentrations of BSME effectively arrested lipid accumulation and inhibited adipocyte maturation, when compared with tea polyphenols. Gene expression level of adipocyte hyperplasia (CEBPα, PPARγ) and lipogenesis (LPL)-related genes have been significantly (p ≤ 0.05) downregulated, and mitochondrial-thermogenesis-associated genes (PPARγc1α, UCP-1, prdm16) have been significantly (p ≤ 0.001) upregulated. The BSME-treated, maturing, adipocyte-secreted proteins were detected with a decreased protein level of leptin, TNF-α, IL-6 and STAT-6, which are associated with insulin resistance and macrophage recruitment. The "LPS-stimulated macrophage" treated with "BSME-treated adipocytes condition media", shown with significant (p ≤ 0.001) decrease in metabolic-inflammation-related proteins-such as PGE-2, MCP-1, TNF-α and NF-κB-were majorly associated with the development of foam cell formation and progression of atherosclerotic lesion. The present findings concluded that the availability of active principles in basil seed effectively inhibit adipocyte hypertrophy, macrophage polarization, and the inflammation associated with insulin resistance and thrombosis development. Ocimum basilicum L. seed may be useful as a dietary supplement to enhance fatty acid oxidation, which aids in overcoming metabolic complications.

Keywords: adipocytes; basil seed; inflammation; lipogenesis; mitochondrial thermogenesis.

Figure 1

GC-MS spectral data for basil seed methanol extract (BSME).

Figure 2

In vitro cytotoxic effect of basil seed methanol extract (BSME) in preadipocytes after 48 h (a) and macrophages after 12 h (b). Results are presented as the mean ± standard deviation (SD) (n = 6 in all the groups).

Figure 3

Determination of lipid accumulation using oil red O staining analysis after basil seed methanol extract (BSME) treatment for 14 days. In oil red O staining, vehicle control showing hypertrophic adipocyte (red arrow) was directly propositional to triglyceride storage. However, in 160 µg/dL of BSME treatment, it showed controlled adipocyte maturation, less lipid accumulation and spindle-shaped (white arrow) adipocytes when compared with 40 and 80 µg/dL of BSME-treated and vehicle control cells.

Figure 4

Effective dose determination of basil seed methanol extract (BSME) on inhibitory potential of lipid accumulation after 14 days treatment. Values are means ± SD (n = 6). * p ≤ 0.05 by comparison with vehicle control. ** p ≤ 0.001 by comparison with vehicle control and tea polyphenol.

Figure 5

Determination of hypertrophic adipocyte and lipid accumulation using Nile red fluorescent staining analysis after basil seed methanol extract (BSME) treatment for 14 days. In Nile red staining, vehicle control showing high red fluorescence (red arrow), directly propositional to accumulation of lipid droplets. However, 160 µg/dL of BSME treatment showed inhibited adipocyte maturation (white arrow) and less lipid accumulation in adipocyte when compared with lower doses of basil-seed-treated maturing adipocytes.

Figure 6

Mitochondrial membrane potential, JC-1 (c) staining images (200×) of vehicle control and basil-seed-methanol-extract-treated (BSME) adipocytes after 14 days. JC-1 fluorescence images showing merged images of the red and green signals of the dye, corresponding to JC-1 in J-aggregates vs. monomeric form. We found less J-aggregates and hypertrophic adipocyte (red arrow) in vehicle control, and in 40 µg/dL and 80 µg/dL of BSME-treated adipocytes. In 160 µg/dL of BSME, showing linear and spindle-shaped adipocytes with high j-aggregates (white arrow), showed high MMP, directly representing active mitochondria.

Figure 7

Effect of basil seed methanol extract (BSME) on adipocyte hyperplasia (a), lipolysis (b) and metabolic-inflammation-related (c) gene expression levels after 14 days. Values are means ± SD (n = 6). * p ≤ 0.05 by comparison with vehicle control and ** p ≤ 0.001 by comparison with vehicle control and tea polyphenol.

Figure 8

Effect of basil seed methanol extract (BSME) on insulin resistance and metabolic-inflammation-related protein (leptin, TNF-α, IL-4 and STAT-6) levels in maturing adipocytes after 14 days. Values are means ± SD (n = 6). * p ≤ 0.05 by comparison with vehicle control and ** p ≤ 0.001 by comparison with vehicle control and tea polyphenol.

Figure 9

Determination of lipid accumulation and macrophage foam cell in basil-seed-treated adipocyte condition media-treated polarized macrophages using oil red O staining analysis after 12 h. Condition media of vehicle control macrophages showed hyper proliferating cells with clumped morphology representing foam cell development (red arrows). Basil seed 40 µg/dL 80 µg/dL and tea polyphenol (160 µg/dL) dose showed a reduced number of proliferating and clumped cells. In 160 µg/dL dose of basil seed showing minimal number of cells, less proliferation and uniform morphology of macrophages (white arrows).

Figure 10

Quantification of metabolic-inflammation-related gene expression levels in basil-seed-methanol-extract-treated (BSME) adipocyte condition media treated macrophage after 12 h. Values are means ± SD (n = 6). * p ≤ 0.05 by comparison with vehicle control. ** p ≤ 0.001 by comparison with vehicle control and tea polyphenol.

Figure 11

Quantification of intracellular protein levels in macrophages treated with basil-seed-treated adipocyte condition media after 12 h. Values are means ± SD (n = 6). * p ≤ 0.05 by comparison with vehicle control. ** p ≤ 0.001 by comparison with vehicle control and tea polyphenol.