Metabolites of oregano (Origanum vulgare) seed and their anti-obesity effects on 3T3-L1 adipocytes through down-regulated adipogenesis

Abstract

Metabolites of the 80% ethanol extract (OSE) and ethyl acetate fraction (OSEA) of oregano seed were analyzed by GC-MS, and anti-obesity effects of OSE and OSEA were evaluated in 3T3-L1 adipocyte. OSE possessed high content of glucose, fructofuranose, and sucrose while OSEA had high content of phenolic chemicals. OSEA contained higher levels of gallic acid, syringic acid, protocatechuic acid, and catechin than OSE. OSEA inhibited lipid droplet accumulation with concentration dependent manner in 3T3-L1 preadipocytes during differentiation. OSEA showed more inhibition ability than OSE by 13.7-fold at the level of 125 μg/mL. Additionally, relative mRNA and protein expression levels of pparγ, c/ebpα, fas, and srebp-1c which are related to adipogenesis were significantly lower in OSEA treatment group than in OSE treatment group (p < 0.05). Therefore, OSEA could be used as anti-obesity functional ingredient.

Keywords: Anti-obesity; Antioxidant; Metabolite; Oregano seed; Spice.

Fig. 1

Multivariate statistical analysis of metabolites. (A) Principle component analysis (PCA) score plot of OSE and OSEA. (B) Partial least square discriminant analysis (PLS-DA) score plot of OSE and OSEA. (C) PLS bi-plot of OSE and OSEA. The triplicate samples of each group are shown in red and the detected metabolites are shown in green. OSE oregano seed 80% ethanol extract, OSEA oregano seed ethyl acetate fraction

Fig. 2

Effect of OSE and OSEA on lipid accumulation in 3T3-L1 adipocytes. (A) Effect of oregano seed 80% ethanol extract (OSE) and oregano seed ethyl acetate fraction (OSEA) on 3T3-L1 preadipocyte viability as measured by MTT assay. (B) Morphology of Oil Red O (ORO) stained adipocytes. Magnification × 200. (C) Optical density values extracted using isopropanol from adipocytes stained with ORO. Pre preadipocyte, AD adipocyte, DM adipose differentiation inducing cocktail (dexamethasone, IBMX, and insulin). The results were expressed as mean ± SEM. MTT assay significant differences between groups (p < 0.05) were identified by Duncan’s multiple range test and indicated by different letters. ORO significant differences between the AD group and each sample treatment group were compared by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Inhibitory effect of OSE and OSEA on adipogenic gene expression. Relative mRNA expression levels of oregano seed 80% ethanol extract (OSE) and oregano seed ethyl acetate fraction (OSEA) groups were analyzed by qRT-PCR. (A) Peroxisome proliferator-activated receptor γ (pparγ). (B) CCAAT Enhancer-binding protein α (c/ebpα). (C) Adipocyte protein 2 (ap2). (D) Cluster of differentiation 36 (cd36). (E) Sterol regulatory element-binding protein 1c (srebp-1c). (F) Acetyl-CoA carboxylase 1 (acc1). (G) Fatty acid synthase (fas). (H) Schematic diagram of OSEA on 3T3-L1 adipocyte differentiation. The relative mRNA expression level of each gene was normalized through the beta-actin value, and the ratio with the adipocyte (AD) group was compared. The experiment was triplicate, and the results were expressed as mean ± SEM. Significant differences between the AD group and each sample treatment group were compared by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

Effect of OSEA on adipogenic protein expression by Western blot analysis. (A) The expression level of adipogenesis related protein. Protein bands in the oreagano seed ethyl acetate fraction (OSEA) treated group measured by Western blot analysis were quantified by Image J program. (B) Peroxisome proliferator-activated receptor γ (PPARγ). (C) CCAAT enhancer-binding protein α (C/EBPα). (D) Fatty acid synthase (FAS). (E) Sterol regulatory element-binding protein 1c (SREBP-1C). (F) Stearoyl-CoA desaturase 1 (SCD1). (G) Adipocyte protein 2 (AP2). The experiment was triplicate, and the results were expressed as mean ± SEM. Significant differences between the adipocyte (AD) group and each sample treatment group were compared by Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)