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.

Introduction

Obesity-related diseases are mainly due to the oxidative stress and proinflammatory adipokines resulting from the accumulation of excessive adipose tissue (Kim et al., 2018; Vekic et al., 2019). As a consequence, risks of getting complications such as insulin resistance, cardiovascular disease, and nonalcoholic fatty liver disease could be high in humans with obesity (Kim et al., 2018; Koliaki et al., 2019; Polyzos et al., 2019; Vekic et al., 2019).

Polyphenolic compounds and terpenoids, which are secondary metabolites in plants are known as phytochemicals with strong antioxidant activity (Graßmann, 2005). Plant-derived polyphenols such as catechin, resveratrol, and curcumin are proven to be effective in reducing inflammation that causes obesity and are reported to inhibit the proliferation and differentiation of adipocytes (Graßmann, 2005). These substances are also known to suppress the expression of genes related to adipocyte proliferation and adipocytic differentiation including peroxisome proliferator-activated receptor γ (pparγ), CCAAT enhancer-binding protein α (c/ebpα), and sterol-regulatory element-binding protein 1c (srebp1c) (Kim et al., 2018; Rayalam et al., 2008). Terpenoids can regulate the activity of PPARs (dietary lipid sensors that control energy homeostasis) and suppress adipogenesis. In addition, terpenoids can regulate the inflammatory response by interfering with the expression of proinflammatory transcription factors (Goto et al., 2010; Rayalam et al., 2008).

Oregano (Origanum vulgare) is an herbal spice native to the Mediterranean and is proven to have antibacterial, antioxidant, and anti-inflammatory effects (Lagouri et al., 1993). In our previous study (Lee et al., 2021), 80% ethanol was employed to extract substances from oregano seed, with fractionation using n-hexane, ethyl acetate, n-butanol, and distilled water, followed by measurement of antioxidant activity. As a result, the highest antioxidant activity and total phenolic content were observed in the oregano seed ethyl acetate fraction (OSEA). In addition, carvacrol, thymoquinone, thymol, and linalool were major volatiles found in OSEA.

The objectives of this study were to evaluate the anti-obesity effects of ethanol extract of oregano seed and OSEA on adipocytic differentiation and to compare the metabolites present in both extracts. Relative mRNA and protein expression levels of transcription factors related to adipocyte proliferation and adipocytic differentiation were analyzed to assess the influence on adipocytic differentiation.

Materials and methods

Materials

n-Hexane, isopropanol, ethanol, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), MTT, dimethyl sulfoxide (DMSO), methoxyamine hydrochloride, pyridine, and Oil Red O (ORO) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Bovine calf serum (BCS), fetal bovine serum (FBS), insulin, trypsin EDTA, and a penicillin–streptomycin solution (P/S) were bought from WELGENE (Gyeongsan, Korea). A formalin solution, RIPA lysis buffer, One-Step Acrylamide Kit, and Tris-buffered saline (TBS) were acquired from Tech & Innovation (Chuncheon, Korea). Tween 20 came from Bio-Rad Laboratories, Inc. (Hercules, CA, USA), and Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Hyclone Laboratories Inc. (Logan, UT, USA). The RT Master Mix and THUNDERBRID® SYBR® qPCR Mix were purchased from TOYOBO (Osaka, Japan), and TRIzol from Invitrogen (Carlsbad, CA, USA). Antibodies against PPARγ, fatty acid synthase (FAS), SREBP-1C, stearoyl-CoA desaturase-1 (SCD1), β-actin (β-ACTIN), adipocyte fatty acid-binding protein (AP2), and C/EBPα (served as primary antibodies) were all bought from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), whereas a horseradish peroxidase-conjugated goat anti-mouse antibody (served as a secondary antibody) was bought from Invitrogen. N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylsilyl chloride and fluoranthene were acquired from Supelco (Bellefonte, PA, USA).

Preparation of the oregano seed 80% ethanol extract (OSE) and OSEA

Oregano seed extracts and fractions including OSE and OSEA were prepared in the same manner as described by Lee et al. (2021). The obtained OSE and OSEA were stored frozen at − 20 °C until analysis.

Metabolite analysis of the OSE and OSEA

Metabolite analysis by GC–MS

Metabolite analyses of the OSE and OSEA were conducted at Seoul National University National Instrumentation Center for Environmental Management (Seoul, Korea). For the metabolite analysis and quantification, the method of Kim et al. (2021) was utilized with minor modifications. For GC–MS analysis of the OSE and OSEA, 50 µL of pyridine containing 20 mg/mL methoxyamine hydrochloride was added to 3 mg of an extract sample, followed by reaction at 30 °C for 90 min. After that, 50 µL of a solution of N,O-bis(trimethylsilyl)trifluoroacetamide and 1% trimethylsilyl chloride was added for trimethylsilyl derivatization, and 30 µL of 500 µg/mL fluoranthene was added as an internal standard material and reacted at 60 °C for 30 min. Gas chromatography (GC; Trace 1310, Thermo Fisher Scientific, Waltham, MA, USA) analysis was performed on 1 µL of a sample; a DB-5MS (60 m × 0.25 mm, 0.25 µm, Agilent Technologies Inc., Santa Clara, CA, USA) column served as a stationary phase, and helium at 1.5 mL/min was used as a mobile phase. The temperature was initially maintained at 50 °C for 2 min, then at a rate of 5 °C/min, it was raised to 180 °C, kept there for 8 min; next, at a rate of 2.5 °C/min, the temperature was raised to 210 °C, and then again at a rate of 5 °C/min to 320 °C, where it was kept for 10 min. During mass spectrometry (MS; ISQ LT single quadrupole Mass Spectrometer, Thermo Fisher Scientific), the injector temperature was maintained at 300 °C, and the transport line and ion source temperatures were kept at 310 °C and 270 °C, respectively. The mass range was set to m/z 35–650, and electron ionization was applied (70 eV).

Metabolite detection and quantification

The retention time and mass spectrum data of each peak obtained by GC–MS were analyzed using a mass spectra library (NIST, v.2.0d, National Institute of Standards and Technology, Gaithersburg, MD, USA). All analyzed metabolites were detected with a similarity of 70% or more to a standard mass spectrum found in the NIST database. Each compound was quantified against the fluoranthene standard.

Inhibitory effects of the OSE and OSEA on 3T3-L1 cell adipogenesis

3T3-L1 preadipocyte culture

The 3T3-L1 preadipocyte line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in the DMEM medium containing 10% of BCS and 1% of P/S in a humidified incubator at 5% CO₂ and 37 °C. Subculturing of 3T3-L1 preadipocytes was conducted at intervals of 2–3 days (Kim et al., 2017).

MTT assay

MTT assay was performed to evaluate the toxicity of OSE and OSEA samples to 3T3-L1 preadipocytes (Cole, 1986). The 3T3-L1 preadipocyte was cultured in a 96-well plate, and an extract sample was added after 48 h, and then the cells were cultured for 1 day. Next, 0.5 mM MTT (in PBS) was added into each well and reacted for 4 h in the CO₂ incubator at 37 °C. Thereafter, the supernatant was removed from the plate wells and replaced with DMSO, and absorbance was measured at 540 nm wavelength using a microplate reader (Eon, BioTek, Winooski, VT, USA).

Adipocytic differentiation of 3T3-L1 preadipocytes

To induce adipogenesis of 3T3-L1 preadipocytes, the cells were seeded in a plate and cultured until confluence. Two days after confluence, the medium was replaced with DMEM containing 10% of FBS and 1% of P/S. Subsequently, the cells were treated with DMI (1 μM dexamethasone, 0.5 mM IBMX, and 5 μg/mL insulin), which is a mixture for the induction of adipogenesis (Zebisch et al., 2012). At the same time, OSE and OSEA were applied at a concentration of 75, 100, or 125 μg/mL. After 48 h of differentiation induction, the medium was replaced with DMEM containing 10% of FBS, 1% of P/S, and 5 μg/mL insulin. Next, the medium was refreshed every 2 day, and an OSE or OSEA sample was added into the medium each time. The differentiation was implemented for 8 days.

ORO staining

After 8 days of adipocytic differentiation of 3T3-L1 cells, the medium was removed, and the cells were washed with PBS. Thereafter, the cells were treated with a 10% formalin solution and stirred at room temperature for 15 min to fix them; then the cells were stained with a 0.5% ORO solution for 30 min. The stained lipid droplets were examined under a microscope (CKX41, OLYMPUS, Tokyo, Japan). Then, each well with cells was incubated with the same amount of isopropanol, and absorbance was measured at 540 nm wavelength using a microplate reader (BioTek) (Choi et al., 2013).

RNA extraction and quantitative real-time PCR (qRT-PCR)

For qRT-PCR analysis, the method of Lee et al. (2020) was utilized. To extract total RNA from differentiated 3T3-L1 adipocytes, the cells were homogenized with the TRIzol reagent. Extracted total RNA was reverse-transcribed into cDNA using the RT Master Mix. Amplification of genes from the synthesized cDNA was performed by qPCR (QuantStudio 3, Applied Biosystems, Foster City, CA, USA) after we mixed the cDNA, oligonucleotide primers for a specific gene, and the THUNDERBRID® SYBR® qPCR Mix. A total of 40 cycles were performed, and the 2^ΔΔCt method was used to determine relative expression levels, which were compared between the control group and extract treatment groups. The oligonucleotide primer sequences were as follows: pparγ 5′-CCATTCTGGCCCACCAAC-3′ (forward), 5′-AATGCGAGTGGTCTTCCATCA-3′ (reverse); c/ebpα 5′-GCGGGCAAAGCCAAGAA-3′ (forward), 5′-GCGTTCCCGCCGTACC-3′ (reverse); fas 5′-GCTGCTGTTGGAAGTCAGC-3′ (forward), 5′-AGTGTTCGTTCCTCGGAGTG-3′ (reverse); srebp-1c 5′-GGTTTTGAACGACATCGAAGA-3′ (forward), 5′-CGGGAAGTCACTGTCTTGGT-3′ (reverse); acetyl-CoA carboxylase 1 (acc1) 5′-GCAGCCCTGGGCACAG-3′ (forward), 5′-GGGAATACCCGTGGGAGTAGTT-3′ (reverse); cluster of differentiation 36 (cd36) 5′-GGCCAAGCTATTGCGACAT-3′ (forward), 5′-CAGATCCGAACACAGCGTAGA-3′ (reverse); ap2 5′-AGCTGGTGGTGGAATGTGTTATGA-3′ (forward), 5′-ATTTCCATCCAGGCCTCTTCCT-3′ (reverse); and β-actin 5′-CTAGGCACCAGGGTGTGATG-3′ (forward), 5′-GTCCCAGTTGGTAACAATGCC-3′ (reverse).

Protein extraction and Western blot analysis

For protein extraction from differentiated 3T3-L1 adipocytes, the cells were lysed using RIPA lysis buffer. Then, the protein concentration in the lysate was quantified with the BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins in the quantified samples were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). Next, TBST was prepared by mixing TBS and 0.2% of Tween 20. Using this buffer, a 5% skim milk solution (Bioshop Canada Inc., Burlington, ON, Canada) was prepared. Using the 5% skim milk, the membrane was blocked at room temperature for 2 h. Thereafter, the membrane was incubated with primary antibodies: anti-PPARγ (1:500 dilution), anti-FAS (1:3000), anti-SREBP-1C (1:500), anti-SCD1 (1:200), anti-AP2 (1:20,000), and anti-C/EBPα (1:200) in the 5% skim milk solution used for blocking, at 4 °C overnight. After washing the membrane with TBST, the membrane with the secondary antibody (1:5000, Invitrogen) was stayed at room temperature for 2 h. Protein signals were developed on X-ray film (CP-BU New, Agfa-Gevaert N.V., Mortsel, Belgium) using the ECL Western Blotting Substrate (SuperSignal™, Thermo Fisher Scientific). Finally, protein amounts in bands were measured by densitometry in ImageJ (LOCI, University of Wisconsin, Madison, WI, USA) and normalized to β-actin.

Statistical analysis

All experiments were conducted three times independently. Results from metabolite analysis and anti-obesity using 3T3-L1 cells were expressed as mean ± standard deviation (SD) and mean ± standard error of the mean (SEM), respectively. Statistical significance of differences between the control group and an extract treatment group was evaluated in SPSS Statistics (v.23, IBM Co., Armonk, NY, USA) by Student’s t test at p < 0.05. In addition, groups were compared by one-way analysis of variance at p < 0.05, and then the significance was verified by Duncan’s multiple-range test. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were performed in SIMCA (v.15, Umetrics, Umeå, Sweden) between groups for the metabolite analysis.

Results and discussion

Metabolite profiling of the OSE and OSEA by GC–MS

The results of metabolite analysis in the OSE and OSEA are shown in Table 1. All 67 substances were detected in OSE, whereas 47 compounds were found in OSEA. The detected substances were categorized into aldehydes, amino acids, carbohydrates, carboxylic acids, flavonoids, fatty acids, phenolic acids, other phenols and other classes. One aldehyde, 5 amino acids, 22 carbohydrates, 15 carboxylic acids, 3 fatty acids, 1 flavonoid, 7 phenolic acids, 8 other phenols and 5 other substances were identified. In the OSE, the ratio of carbohydrates to amino acids was higher than that in the OSEA. Among all the detected substances, OSE had significantly higher levels of glucose, fructofuranose and sucrose than OSEA (p < 0.05). In the OSEA, the content of phenolic acids and flavonoid group was higher than those in the OSE, considering all the detected substances. The levels of caffeic acid, catechin, and protocatechuic acid in OSEA showed a significantly higher than those in OSE (p < 0.05). Additionally, the content of substances having antioxidant activity such as gallic acid, syringic acid, and vanillic acid was significantly higher in the OSEA than in the OSE (p < 0.05). Coccimiglio et al. (2016) reported that an oregano (O. vulgare) ethanol extract contains abundant monoterpene hydrocarbons and phenolic compounds showing high antioxidant activity. Tsimogiannis et al. (2006) performed GC–MS analysis on an oregano (O. heracleoticum) extract and found out that the ethanol extract contained glycosides and free sugars, whereas the ethyl acetate extract had compounds of intermediate polarity.

Table 1.

Oregano seed metabolites between OSE and OSEA detected by GC–MS

Compounds	RT (min)	OSE	OSEA	Ion	TMS
		(Fluoranthene equivalent mg/g extract)
Aldehyde
4-Hydroxybenzaldehyde	26.2	5.6 ± 2.8	44.4 ± 7.3***	267	2
Amino acids
Valine	21.6	133.3 ± 5.3**	ND	144	2
Pyroglutamic acid	30.0	310.0 ± 3.1***	3.3 ± 0.3	156	2
γ-Aminobutyric acid	30.2	93.3 ± 2.6***	ND	174	3
Alanine	18.2	221.1 ± 14.7***	ND	116	2
l-Proline	24.0	87.8 ± 2.3*	ND	142	2
Carbohydrates
Glyceryl glycoside	58.7	71.1 ± 15.2***	ND	204	6
Glyceric acid	26.2	41.1 ± 8.1**	3.3 ± 0.1	189	3
Myo-Inositol	53.0	931.1 ± 50.6***	ND	217	6
Ribitol	37.6	92.2 ± 25.5***	ND	217	5
Mannitol	47.0	177.8 ± 32.1***	ND	319	6
Xylitol (1)a	36.3	237.8 ± 30.1***	ND	147	5
Xylitol (2)	37.4	42.2 ± 2.8***	ND	217	4
l-Threitol	29.2	20.0 ± 1.2***	ND	217	4
Galactinol	57.4	98.9 ± 29.3***	ND	204	9
d-Ribose	38.4	430.0 ± 55.5***	24.4 ± 1.8	217	4
β-d-Glucopyranose	49.2	10.0 ± 2.5	38.9 ± 4.5***	204	5
d-Glucose	49.3	10,215.6 ± 112.4***	36.7 ± 6.2	204	5
Gulose	52.3	247.8 ± 3.5*	ND	204	5
Fructofuranose (1)	47.8	6998.9 ± 530.1***	ND	217	5
Fructofuranose (2)	41.4	2534.4 ± 51.7***	ND	204	5
Fructopyranose	42.0	4151.1 ± 191.9***	ND	217	5
Ribofuranose (1)	39.6	114.4 ± 36.9***	44.4 ± 6.3	217	4
Ribofuranose (2)	40.1	274.4 ± 22.6***	10.0 ± 3.0	217	4
α-dl-Arabinopyranose	34.1	27.8 ± 2.3***	ND	191	4
Sucrose	64.6	8708.9 ± 227.5***	3.3 ± 0.4	361	8
Melibiose	71.6	188.9 ± 10.7**	13.3 ± 5.3	204	8
Trehalose	87.7	376.7 ± 27.1	521.1 ± 32.1	361	8
Carboxylic acids
Malonic acid	21.2	341.1 ± 17.1***	10.0 ± 4.1	147	2
Succinic acid	24.4	455.6 ± 62.3**	177.8 ± 9.5	147	2
l-Threonic acid (1)	30.5	1387.8 ± 143.4**	30.0 ± 6.1	292	4
l-Threonic acid (2)	31.0	34.4 ± 3.3**	ND	292	4
Glycolic acid	17.3	104.4 ± 32.7	171.1 ± 8.8**	147	2
Hexenoic acid	18.8	4.4 ± 1.0	17.8 ± 1.9**	171	1

Compounds	RT	OSE	OSEA	Ion	TMS
5-Indolecarboxylic acid	50.7	7.8 ± 1.7	45.6 ± 10.4***	246	2
β-Lactate	19.4	144.4 ± 62.6*	70.0 ± 14.7	147	2
Lactic acid	16.8	148.9 ± 66.5	223.3 ± 35.0*	147	2
Malic acid	28.9	396.7 ± 47.2***	ND	147	3
2-Hydroxybutyric acid	18.9	3.3 ± 0.3	20.0 ± 1.2***	131	2
2-Deoxytetronic acid	26.2	5.6 ± 3.0	44.4 ± 6.1***	189	3
ρ-Coumaric acid	47.4	21.1 ± 1.3	36.7 ± 4.0**	293	2
Azelaic acid	41.0	10.0 ± 1.0	274.4 ± 22.8***	317	2
3,4-Dihydroxyphenylacetic acid	42.4	21.1 ± 1.3	55.6 ± 9.3***	179	2
Fatty acids
Linolenic acid	56.3	4.4 ± 0.6	3.3 ± 1.1	337	1
Palmitic acid	51.6	47.8 ± 6.9	88.9 ± 23.7*	117	1
Stearic acid	57.1	6.7 ± 1.8	22.2 ± 2.0***	341	1
Flavonoids
Catechin	69.4	158.9 ± 7.6	1194.4 ± 42.8***	368	5
Phenolic acids
Gallic acid	48.2	3.3 ± 1.1	10.0 ± 3.0***	281	4
Benzoic acid	22.6	14.4 ± 6.2	50.0 ± 7.5***	179	1
Vanillic acid	39.5	14.4 ± 6.2	58.9 ± 10.4***	267	2
Gentisic acid	29.7	24.4 ± 4.9	121.1 ± 14.8***	267	2
Protocatechuic acid	42.0	142.2 ± 17.7	408.9 ± 50.2***	193	3
Caffeic acid	76.6	2427.8 ± 132.9	10,442.2 ± 192.0**	396	5
Syringic acid	45.5	10.0 ± 3.0	38.9 ± 4.5***	327	2
Other phenols
Catechollactate	52.1	2071.1 ± 128.7**	697.8 ± 14.6	267	4
4-Hydroxy benzene methanol	29.5	54.4 ± 8.9	166.7 ± 8.3***	197	2
Thymol	24.4	344.4 ± 12.5	84.4 ± 8.1	207	1
tert-Butylhydroquinone (1)	29.6	72.2 ± 5.4	157.8 ± 14.3**	310	2
tert-Butylhydroquinone (2)	31.2	248.9 ± 18.6	302.2 ± 27.4**	295	2
Isoeugenol	28.6	213.3 ± 13.7***	54.4 ± 8.9	206	1
4-tert-Butylcatechol	31.6	63.3 ± 6.9	345.6 ± 18.2***	295	2
1,4-Dihydroxybenzene	26.8	68.9 ± 2.8	133.3 ± 17.5***	239	2
Others
Borneol	22.2	41.1 ± 5.1*	13.3 ± 5.3	95	1
Ethyl phosphoric acid	21.8	24.4 ± 1.8***	ND	211	2
Ethylene glycol	14.5	484.4 ± 4.8*	336.7 ± 4.9	147	2
Glycerol	23.2	1387.8 ± 57.8**	30.0 ± 2.7	147	3
Phosphate	23.1	64.4 ± 12.5	47.8 ± 6.9	299	3

The results were expressed as mean ± SD. Significant differences between OSE and OSEA were compared with Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

RT retention time; OSE oregano seed 80% ethanol extract; OSEA oregano seed ethyl acetate fraction; Ion: quantitative ion; TMS trimethylsilylation; Level mean values (n = 3) and fluoranthene equivalent mg/g extract; ND not detected

^aA chemical can show a little different ion fragment patterns from derivatization process, which elute different retention time

The PCA score plots of the substances detected by GC–MS in the OSE and OSEA are shown in Fig. 1A. PC1 and PC2 (principal components) explained 92.8% and 5.1% of variance, respectively. Next, PLS-DA was performed based on the PCA results, and the score plot and bi-plot results are presented in Fig. 1B and C, respectively. The score plot and bi-plot of PLS-DA indicated the degree of separation of substances between OSE and OSEA by subdividing each group for the OSE and OSEA into components. Differences between the OSE and OSEA groups turned out to be subdivided in a manner similar to that in PCA (Fig. 1B, C). PLS component 1 and PLS component 2 were responsible for 92.8% and 5.0% of variance, respectively, and 97.8% in total, indicating that the substances present in OSE and OSEA separated the two types of extracts effectively. Parameters R2X, R2Y, and Q2 (cum) for PLS-DA modeling were 0.998, 1.0, and 0.999, respectively, and the R2 and Q2 values for 100 permutations were 0.964 and 0.423, respectively.

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

The correlation between the OSE and OSEA in terms of major substances is given in Table 2. The major metabolites in the OSE were d-glucose and sucrose. On the other hand, the major metabolites in the OSEA were gallic, gentisic, vanillic, syringic, and protocatechuic acids. Zhang and de Mejia (2020) prepared extracts of Cucumis melo L. using different solvent systems and evaluated anticancer activity and metabolite profiling analysis. The yield of polyphenols in the extract was affected by the extraction solvent, and that the anticancer activity increased depending on the solubility of chemicals in the solvent (Zhang and de Mejia, 2020).

Table 2.

Component correlation matrix of OSE and OSEA by PLS-DA

Group	Compounds	Correlation matrix
OSE	d-Glucose	1.000
	Sucrose	0.990
	d-Ribose	0.990
	Myo-Inositol	0.987
	Ribitol	0.985
	Fructopyranose	0.984
	Ethyl phosphoric acid	0.984
	Pyroglutamic acid	0.983
	Xylitol	0.983
	Fructofuranose	0.982
	l-Threitol	0.982
	Glyceric acid	0.981
	Malic acid	0.980
	Ribofuranose	0.979
	α-dl-Arabinopyranose	0.978
	Galactinol	0.978
	Alanine	0.977
OSEA	Gallic acid	1.000
	Azelaic acid	1.000
	2-Hydroxybutyric acid	1.000
	Gentisic acid	1.000
	β-d-Glucopyranose	0.998
	Vanillic acid	0.998
	Syringic acid	0.997
	Oxitriptan	0.997
	Protocatechuic acid	0.997
	1,4-Dihydroxybenzene	0.997
	4-Hydroxybenzoic acid	0.995
	Benzoic acid	0.994
	Catechin	0.993
	Caffeic acid	0.987
	tert-Butylhydroquinone	0.984
	Hexenoic acid	0.973
	Glycolic acid	0.967

OSE oregano seed 80% ethanol extract; OSEA oregano seed ethyl acetate fraction

In the metabolite analysis, the OSE contained a high proportion of carbohydrates and amino acids, whereas the OSEA had a high proportion of carboxylic acid families. Moreover, the OSE and OSEA possessed substances known to have antioxidant activity, such as gentisic acid, vanillic acid, syringic acid, protocatechuic acid, and catechin, whereas the OSE contains significantly lower concentrations of such substances than the OSEA does (p < 0.05). Therefore, OSE and OSEA possessed different the types and levels of metabolites.

Effects of OSE and OSEA on adipocytic differentiation

Results of MTT assay and ORO staining are shown in Fig. 2. In both OSE and OSEA treatment groups, no significant changes were observed in cell viability in the 25–100 μg/mL concentration range (p < 0.05) while cell viability in the OSE and OSEA groups at the 150 μg/mL was 77.9% and 75.8%, respectively (Fig. 2A). Both the OSE and OSEA suppressed the formation of fat globules and decreased the number of stained lipid droplets. The OSEA treated group showed a significant decrease in lipid droplet accumulation compared to OSE treated group. At 75, 100, and 125 μg/mL, the OSE yielded 105.8%, 82.9%, and 77.8% lipid accumulation, respectively compared to the adipocyte [AD] group, whereas the OSEA produced significant differences in lipid accumulation of 79.0%, 41.9%, and 5.7%, respectively (p < 0.05). Overall, the OSEA has a stronger inhibitory effect on lipid accumulation than the OSE does.

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)

The accumulation of body fat is proportional to the increase in the number and size of adipocytes, and phytochemicals such as phenolic compounds and terpenoid compounds abundant in plant extracts, control the cellular metabolism of preadipocytes and inhibit their differentiation into adipocytes (Seo et al., 2015). John and Arockiasamy (2021) studied the reduction of reactive oxygen species in 3T3-L1 adipocytes by syringic acid and found out syringic acid significantly raised the levels of intracellular antioxidant enzymes such as superoxide dismutase and catalase and can help reduce the stress caused by reactive oxygen species. Furthermore, Zhang and de Mejia (2020) analyzed the relation between protocatechuic acid and adipogenesis-induced inflammation signs in 3T3-L1 adipocytes. Protocatechuic acid can inhibit secretion of proinflammatory cytokines from adipocytes and increase the secretion of anti-inflammatory cytokines. In this way, strong anti-obesity activity of phytochemicals could be due to the suppressed accumulation of excessive lipids (Abdali et al., 2015; Sun et al., 2016).

The metabolite analysis revealed that the OSE and OSEA had different profiles of substances. Phenolic compounds and terpenoids known to have antioxidant activity were detected in larger amounts in the OSEA than in OSE. The observed difference in lipid accumulation in 3T3-L1 adipocytes in our experiments could be due to differences in the type and levels of substances having such a physiological activity.

Influence of OSE and OSEA on adipogenic gene expression

Measurement of relative mRNA expression levels of genes involved in 3T3-L1 adipocytic differentiation are depicted in Fig. 3. Pparγ (Fig. 3A) was proved to be significantly downregulated (by 1.4-fold) in the OSE group at the 125 μg/mL compared to the AD group (p < 0.05). The OSEA group at 75, 100, and 125 μg/mL downregulated the expression of this gene significantly by 1.8-, 4.3-, and 25.0-fold, respectively (p < 0.05). C/ebpα (Fig. 3B) expression in the OSE group was not significantly different from its level in the AD group at all concentrations whereas the OSEA at the concentrations of 100 and 125 μg/mL significantly decreased this gene’s expression by 7.1- and 33.3-fold, respectively (p < 0.05). Regarding cd36 and ap2 (Fig. 3C, D), the OSE group showed no significant difference from the AD group at all concentrations. On the contrary, in the OSEA group, there was a significant difference: a concentration-dependent decrease in expression at all concentrations (p < 0.05). Cd36 was significantly downregulated by 1.3-, 2.7-, and 33.0-fold at OSEA concentrations of 75, 100, and 125 μg/mL, respectively. In addition, ap2 was significantly downregulated by the OSEA by 1.9-, 4.6-, and 50.0-fold, respectively (p < 0.05). In the case of srebp-1c (Fig. 3E), no significant difference was found between the OSE group and AD group, while the OSEA at 100 and 125 μg/mL significantly reduced its expression by 2.0- and 4.0-fold, respectively (p < 0.05). In the case of acc1 (Fig. 3F), there was no significant difference between the OSE group and AD group. On the other hand, the OSEA at 100 and 125 μg/mL significantly decreased its expression, by 1.9- and 3.4-fold, respectively (p < 0.05). No significant difference was found in expression of fas between the OSE group and AD group (Fig. 3G) whereas the OSEA at 100 and 125 μg/mL significantly decreased this gene’s expression, by 6.6- and 12.5-fold, respectively (p < 0.05).

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)

PPARγ, which is a gene mainly active in fat tissue, promotes the formation of adipocytes, lipid accumulation, and adipokine production and regulates insulin sensitivity by stimulating fat formation and lipid accumulation in the liver and by upregulating glucose transporter 4 (GLUT4) in muscles (Evans et al., 2004; Yamauchi et al., 2001). Therefore, the PPARγ protein acts as a master regulator of the homeostasis of lipid and glucose metabolism in the liver, muscles, and adipose tissue and controls the expression of target genes that promote adipogenesis, lipid storage, and inflammation, e.g., aP2 and Cd36 (Lim et al., 2006; Makowski et al., 2005). According to studies by Wu et al. (1998) and Furuyashiki et al. (2004), c/ebpα is activated during the differentiation process together with pparγ, and overexpression of both genes induces and accelerates the differentiation into adipocytes. Besides, the promotion of adipogenesis in 3T3-L1 cells is due to the activation of two genes: c/ebpα and pparγ (Shang et al., 2007). Along with c/ebpα, pparγ, and srebp, which modulates adipogenesis, is proven to be involved in cholesterol and fatty acid metabolism. The SREBP-1C protein, an isomer of SREBP, is responsible for the activation of target genes such as fas, acc1, and scd1 encoding fatty acid synthase (Evans et al., 2004; Lee et al., 2010).

Figure 3H is a schematic diagram of transcription factors related to adipogenesis and adipogenesis processes described above and target genes of transcription factors. Adipogenesis represents a process where adipocytes are formed from preadipocytes in the initial fibroblast state via certain transformation. As described above, several transcription factors involved in adipogenesis participate in this phenomenon, and the presence or absence of activation of these transcription factors is a marker that can help to distinguish preadipocytes from adipocytes. At the early stage of differentiation, C/EBPβ stimulates the activation of such transcription factor genes as c/ebpα and pparγ, and the active transcription factors C/EBPα and PPARγ are responsible for late stages of adipocytic differentiation, e.g., induction of target genes ap2, fas, cd36, scd1, and acc1. As for SREBP-1C, it takes part in the synthesis of fatty acids and triglycerides together with C/EBPα and PPARγ.

Therefore, OSEA treatment reduced relative mRNA expression levels of c/ebpα and pparγ, and this alteration seemed to inhibit a series of processes underlying the differentiation of 3T3-L1 preadipocytes into adipocytes. The target genes, ap2 and cd36, are also suppressed due to a decrease in the relative mRNA expression of c/ebpα and pparγ, and this change is thought to inhibit the growth of adipocytes and accumulation of lipids in them. Furthermore, a decrease in the expression of transcription factors that regulate cholesterol and fatty acid metabolism, such as SREBP-1C, can downregulate the relative mRNA expression of SREBP-1C’s target genes, fas and acc1. Thereby inhibiting the enzymatic activity required for fatty acid synthesis. As a result, it is possible to repress adipocyte proliferation and differentiation of 3T3-L1 preadipocytes and to decrease the rate of differentiation into adipocytes via the suppression of mRNA expression levels of c/ebpα and pparγ.

Impact of OSE and OSEA on adipogenic-protein expression

Because the OSEA only significantly reduced relative mRNA expression levels of genes related to adipogenesis, OSEA was analyzed further for adipogenic-protein expression. The processed protein expression data are shown in Fig. 4A. The density of the bands of PPARγ, C/EBPα, FAS, SREBP-1C, and AP2 was lower in the OSEA 125 μg/mL treatment group than in the AD group, thereby confirming that the expression of proteins related to adipogenesis decreased. In particular, the protein band density of SCD1 was reduced when the concentration of OSEA increased from 75 to 125 μg/mL in a concentration-dependent manner compared to the AD group.

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)

The amount of the expressed protein was measured by densitometry and normalized to β-actin, and the quantified results are displayed in Fig. 4. The protein expression levels of PPARγ (Fig. 4B), C/EBPα (Fig. 4C), and SREBP-1C (Fig. 4E) were significantly lower in the OSEA 125 μg/mL treatment group than in the AD group by 5.9-, 1.5-, and 2.6-fold, respectively (p < 0.05). In the case of FAS (Fig. 4D), protein expression was significantly lower in the OSEA 100 and 125 μg/mL treatment groups than in the AD group by 1.5- and 4.7-fold, respectively (p < 0.05). As for SCD1 (Fig. 4F) and AP2 (Fig. 4G), the protein expression decreased by the OSEA (compared to the AD group) in a dose-dependent manner at all concentrations. SCD1 protein expression in OSEA 75, 100, and 125 μg/mL treatment group was significantly decreased, by 1.9-, 12-, and 13.8-fold, respectively, and AP2 protein expression was significantly decreased, by 1.3-, 1.9-, and 6.4-fold, respectively (p < 0.05). Results of protein expression study revealed a trend similar to those of relative mRNA expression levels, indicating that the OSEA effectively reduced both mRNA expression and amounts of the proteins related to adipocytic differentiation and lipid accumulation.

In conclusion, metabolite analysis of OSEA possessed higher content of phenolic compounds including gallic, gentisic, vanillic, syringic and protocatechuic acids than OSE, which possessed substantial amount of glucose and sucrose. Phenolic and terpenoid compounds in OSEA may participate in the suppressive effect on mRNA expression and the proteins related to adipocytic differentiation and lipid accumulation. The results of this study extend the application areas of oregano seeds possessing anti-obesity effects.

Declarations

Conflict interest

The authors declare no conflict of interest.