Amelioratory Effects of Acorn Mook Ethanol Extract on ROS Production and Lipid Accumulation in Differentiated 3T3-L1 Cells

1. Preparation of Samples

Dried whole acorns without outer peel were purchased from Cholokmayl Co. (Seoul, Korea). These acorns were lyophilized acorn and ground into powder. Acorn powder was then extracted with 70% aqueous ethanol (1:10, w/v) by vortexing for two hours at room temperature followed by centrifugation at 10,620 g for 10 min. After filtering ethanol acorn extract (AEtOH) through a 0.22 μm PIFE filter unit, the extract was then concentrated using a rotary vacuum evaporator (N-1110, EYELA, Tokyo, Japan). Samples were then freeze-dried by storing in a deep freezer (WUF-500; Daihan Scientific Co., Gangwon, Korea). They were stored until anti-oxidation analysis and cell culture.

2. Antioxidant Activities of AEtOH

Total polyphenol content of acorn ethanol extract (AEtOH) was analyzed with the method described by Gutfinger (Gutifinger T & Letan A 1974) with slight modifications. Briefly, 50 μL of 2 N Folin-Ciocalteu reagent was added to 500 μL of extract. The mixture was left at room temperature for 3 minutes. Then 500 μL of 20% Na₂CO₃ solution was added to the mixture followed by incubation at 25℃ for 1 hour. Thereafter, 100 μL of this mixture was collected for ELISA. Absorbance was measured at wavelength of 725 nm on Tecan Infinite M200Pro plate reader (Green Mate Bio, Seoul, Korea). The absorbance value was used to calculate total polyphenol content by creating a standard curve with tannic acid (TAE). Total polyphenol content is expressed as mg TAE/g. Total flavonoid contents of acorn ethanol extract (AEtOH) was measured by Moreno MI et al (Moreno MI et al 2000). Briefly, sample solution (0.5 mL) was mixed with 1.5 mL ethanol (95%, v/v), 0.1 mL of aluminum chloride (10%, w/v), 0.1 mL of potassium acetate (1 M), and distilled water. After incubation at room temperature for 30 min, absorbance was determined at 415 nm. A calibration curve was prepared using rutin. Results are expressed as mg of rutin equivalents (mg RE/g). Antioxidant scavenging activity was measured using 1, 1-diphenyl,2-picrylhydrazyl free radical (DPPH). Various concentration of test solution in 0.1mL was added to 0.9 mL of 0.1 mM of DPPH solution in ethanol. Only ethanol (0.1 mL) was used as experimental control. After 30 min of incubation at room temperature, absorbance was measured at wavelength of 517 nm. The scavenging activity of the sample corresponded to the intensity of quenching DPPH. Percent scavenging activity was computed using the following equation:

ABTS radical cations were produced by reacting ABTS (7 mM) and potassium persulfate (2.45 mM) at room temperature in the dark for 16 hours. The solution obtained was further diluted with PBS to give an absorbance of 1.000. Test sample (50 μL) at different concentrations was then added to ABTS working solution (950 μL) to give a final volume of 1 mL. Absorbance was recorded immediately at 734 nm (Auddy B et al 2003).

For ORAC assay, an automated plate reader (KC4, Bio Tek, USA) and 96-well plates were used (Prior RL et al 2003). Assays were conducted in phosphate buffer (pH 7.4) at 37℃. Peroxyl radical was generated using 2,2’-azobis (2-amidino-propane) dihydrochloride prepared fresh for each run. Fluorescein was used as substrate. Fluorescence conditions were as follows: excitation, 485 nm; emission, 520 nm. The standard curve was linear between 0 and 50 μM of Trolox. Results are expressed as μM TE/g fresh mass.

3. Cell Culture and Differentiation

3T3-L1 preadipocytes were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). They were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, Thermo scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone Laboratories INC, Gibco, NY, USA) and 1% (w/v) penicillin-streptomycin at 37℃ in a humidified 5% CO₂ atmosphere incubator (Thermo scientific, Waltham, MA, USA). Two days later, confluent cells were placed in differentiation medium containing 1 μg/mL insulin (I9278, Sigma, St. Louis, MO, USA), 0.5 mM IBMX [3-isobutyl–1-methyl xanthine (I7018, Sigma), and 1 μM dexamethasone (D4902, Sigma). The medium was subsequently replaced with fresh DMEM medium with 1 μg/mL insulin (I9278, Sigma). It was replaced again with fresh DMEM medium containing 10% (w/v) FBS.

4. Cell Cytotoxity

3T3-L1 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and cultured at 37℃ under 5% CO₂ for 24 h. Cells were then treated with different concentrations (0, 100, 300, and 500 μg/mL) of AEtOH extract. Following incubation for 24 h, cells were mixed with MTT solution (120 μL/well) and cultured for 4 h. Cell viability was determined based on difference in absorbance at wavelength of 540 nm (BN 02514, Molecular Device, Toronto, Canada).

5. Western Blotting Analysis

AEtOH treated 3T3-L1 cells were washed twice with ice-cold PBS and harvested with 0.25% (w/v) trypsin/EDTA on day 8. To prepare whole-cell extracts, cells were lysed in 50 μL of cell lysis buffer for 30 min on ice. Protein concentration of cell lysate was determined with Bradford method (Bradford MM 1976). Equal amounts of protein were subjected to 12% (w/v) SDS-polyacrylamide gel electrophoresis (Mini Protein Tetra Cell; Bio-Rad) and transferred onto nitrocellulose membrane (Mini Trans-Blot Transfer Cell; Bio-Rad) which was washed once with washing buffer (TBST, 0.1% (v/v) Tween 20). After blocking with a blocking buffer (PBS, 5% (w/v) skim milk, 0.05% (v/v) Tween 20) for 1 h, membranes were incubated with primary antibodies for 8 h at 4℃. Mouse polyclonal antibodies (C/ EBP-β, PPAR-γ, C/EBP-α, aP2, Santa Cruz) were diluted 1:1,000. Mouse polyclonal anti-β-actin (sc-47778, Santa Cruz) was diluted 1:2,000 in blocking buffer. After incubation, membranes were washed twice with washing buffer and subsequently incubated with goat anti-rabbit IgG HPR-conjugated secondary antibody (sc-2004; Santa Cruz, diluted 1:5,000 in blocking buffer) at room temperature for 1 h. After washing membranes twice with washing buffer, protein bands were detected using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech; Buckinghamshire, UK) and exposed to X-ray film (Eastman Kodak; Rochester, NY, USA). Band intensities were quantified with Scion-Image for Windows (National Institutes of Health, Bethesda, MD, USA).

6. Acorn Mook Processing and Preparation of Acorn Mook Ethanol Extracts

Ingredients of acorn mook were: 40 g of dried whole acorn (Quercus acutissima Carr.) powder, 60 g of mung bean starch, 3 g of salt, and 900 mL of water. Acorn mook was processed as shown in Fig. 1 and 2. All ingredients were mixed together and soaked for 3 hrs at room temperature (stage 1). Soaked ingredients were boiled at 100℃ for 8 min and heated at 80℃ for 20 min (stage 2). Finally, acorn mook product was poured into a 15 cm × 15 cm × 4.5 cm square bottle, left at room temperature for 1 hr, and then left at 4℃ for 2 hrs (stage 3). After processing of acorn mook, acorn mook ethanol extract was prepared by the same method used for acorn ethanol extraction.

Fig. 1.

Acorn mook and ingredients. Ingredients of acorn mook (AM) were 40 g of dried whole acorn (Quercus acutissima Carr.) powder, 60 g of mung bean starch, 3 g of salt, and 900 mL of water.

Fig. 2.

Acorn mook making process. Acorn mook was prepared as shown in Fig. 1.

7. ROS Production

Cellular ROS generation was measured using 2’,7’-dichlorofluorescein diacertate (DCF-DA, Sigma) as described before (Kim HJ et al 2015). AEtOH or AMEtOH-treated (0, 100, 300, and 500 μg/mL) cells were seeded into 96-well plates at density of 2 × 10⁵ cells/mL and cultured. DCF-DA was added to each well and incubated at 37℃ for 45 min. Cells were then washed with phosphate buffered saline (PBS) twice. The absorbance of each well was then measured at 495 nm/527 nm using a microplate reader.

8. Oil-red O Straining

For qualitative and quantitative analysis of intracellular lipids, oil red O staining was performed as described previously (Thomson et al 2004). Briefly, differentiated 3T3-L1 adipocytes treated with AEtOH or AMEtOH at various concentrations (0, 100, 300, and 500 μg/mL) were washed twice with PBS (P6148, Sigma) and fixed with 200 μL of 4% (w/v) formaldehyde at 4℃ for 1 h. After removing fixing solution, cells were stained with 500 μL of Oil-red O solution (60% 2-propanol and 40% water, filtered with a 0.45 μm filter) for 30 min. After removing the staining solution, plates were rinsed with water three times and dried. Stained lipid droplets appeared red under a light microscope equipped with a chargecoupled device (CCD) camera (BX-51, Olympus, Tokyo, Japan). Oil red O-positive cells were also measured with a microplate reader at wavelength of 500 nm.

9. Statistical Analysis

Results are represented as means±standard deviations. To determine differences between groups, data were analyzed by one-way analysis of variance (one-way ANOVA) with Statistical Package for Social Sciences (SPSS) version 21.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was considered at p<0.05.

1. Antioxidant Activities of AEtOH

Total polyphenol, total flavonoid content, DPPH radical scavenging activity, ABTS radical scavenging activity, and ORAC of acorn ethanol extract (AEtOH) were showed to be 476.44±14.70 mg TAE/g, 102.91±13.14 mg QE/g, 88.36%, 75.04%, and 81.51 μM TE/g, respectively (Table 1). Polyphenols is a chemical compound found in plants, such as catechin, resveratrol and quercetin. Flavonoids and isoflavones are also a kind of polyphenols. These polyphenols have antioxidant effects that scavenger reactive oxygen in our body, preventing aging. It is also known to prevent heart disease with anticancer activity and with potential efficacy to modulate obesity and related disorders (Mattera R et al 2017). Therefore, acorn is rich in phenolic compounds and our results suggest that AEtOH has antioxidant properties.

Table 1.

Total polyphenol and total flavonoid contents of AEtOH

2. Cell Cytotoxicity of AEtOH

To determine cell cytotoxicity after exposure to AEtOH, extract was used to treat cells at different concentrations (0, 100, 300, and 500 μg/mL) for 24 h. The cell cytotoxicity was 94% or higher under all conditions (Fig. 3). This shows that AEtOH has no cytotoxic effect on 3T3-L1 cells.

Fig. 3.

Effect of AEtOH extracts on cell viability in 3T3-L1 cells. 3T3-L1 cells were treated with AEtOH extracts at different concentrations (10, 300, and 500 μg/mL) for 24 h. Cell viability was tested using the MTT assay.

3. Inhibitory Effect of AEtOH on C/EBPα, C/EBPβ, PPARγ, and aP2 Protein Expression in Differentiated 3T3-L1 Preadipocytes

Adipose tissue is an endocrine organ that secretes many cytokinenes and regulates preadipose cell growth, adipose differentiation and lipogenesis in fat cells (Khalipourfarshbafi M 2019). During adipocyte differentiation, fibroblast-like preadipocytes differentiate into lipid accumulation and insulinresponsive mature adipocytes. Increased adipocyte cell number or the size of adipocytes leads to fat accumulation (Rizzatti V et al 2013). Adipocyte differentiation rely on coordinated regulation of transcription factors and PPARγ is seemed to a critical transcription factor in adipogenesis. It can induce the expression of C/EBPα. C/EBPα is another critical transcription factor in adipogenesis (Rosen ED 2002). This is consistent with previous finding showing that acorn methanol extracts can inhibit mRNA expression levels of C/EBPα, C/EBPβ, and PPARγ in differentiated 3T3-L1 cells (EL-Jack AK 1999). Therefore, in order to examine the effects of acorn ethanol extracts (AEtOH) on the expression C/EBPα, C/EBPβ, PPARγ, and aP2, 3T3-L1 pre-adipocytes, AEtOH was treated at different (0, 100, 300, and 500 μg/mL) concentrations. Results showed that protein expression levels of C/EBPα, C/EBPβ, PPARγ, and aP2 in adipocytes treated with AEtOH (500 μg/mL) were significantly decreased compared with those in control cells (Fig. 4).

Fig. 4.

Effect of AEtOH on the expression of C/EBP-β, C/EBP-α, PPAR-γ, and aP2 protein in 3T3-L1 cells. 3T3-L1 cells were treated of different concentrations of AEtOH (0, 100, 300, and 500 μg/mL) for 8 days. From whole cell lysate, protein was resolved by 12% SDS-PAGE for determination of C/EBP-β, C/EBP-α, PPAR-γ, and aP2 expression. β-actin expression is shown as a loading control. The results are reported as the mean±S.D. of three independent experiment (n=3). UC: undifferentiated 3T3-L1 preadipocyte cell, Con: differentiated 3T3-L1 preadipocyte cells without treatment of acorn ethanol extract. (A) CCAAT/enhancer binding protein peroxisome-β (C/EBP-β), (B) CAAT/enhancer binding protein peroxisome-α (C/EBP-α), (C) Peroxisome proliferator-activated receptor-γ (PPAR-γ), (D) Adipocyte protein 2 (aP2).Significantly different with compared to control (Con) by student’s t-test (p<0.05).

4. ROS Production and Total Lipid Accumulation in 3T3-L1 Cells

Moreover, I evaluated the influence of acorn mook ethanol extracts (AMEtOH) of ROS production and lipid accumulation. Reactive oxygen species (ROS) are important regulators of eukaryotic signal transduction. They are also dangerous because they can oxidize proteins, lipids, and DNA, thus impairing the function of these molecules (Rhee SG 2006). Increased oxidative stress is a key factor that causes obesity-related metabolic complications. In other words, when the oxidative stress is increased in the adipose tissue, it is induces an inflammatory signals, adipokine dysregulation, and metabolic diseases and thus it is very significant to inhibit the production of reactive oxygen species (Maggei et al 2018). And the main roles of adipocytes in vertebrates include triglycerides or releasing free fatty acids in response to change in energy demands (Keaney JF et al 2003). Therefore the effects of different concentrations of AEtOH and AMEtOH on 3T3-L1 cells on ROS production and fat accumulation were examined.

Results were shown in Fig. 5. ROS production in 3T3-L1 cells treated with AMEtOH at various concentrations (0, 100, 300, and 500 μg/mL) was significantly (p<0.05) inhibited (by 6%, 25%, and 35% at 100, 300, and 500 μg/mL, respectively) compared to that treated with AEtOH. The inhibitory effects of AMEtOH (0, 100, 300, and 500 μg/mL) on adipogenesis of 3T3-L1 cells compared to those of AEtOH were visualized using Oil-red O staining (Fig. 6). The level of lipid accumulation in AMEtOH-treated 3T3-L1 cells was significantly (p<0.05) suppressed (by 20.3% following treatment with 300 μg/mL of AMEtOH and by 37.3% following treatment with 500 μg/mL of AMEtOH) compared to that in AEtOH-treated cells.

Fig. 5.

Effect of AEtOH and AMEtOH on ROS production in 3T3-L1 cells. The results are reported as the mean±S.D. of three independent experiment (n=3). Values with different super-script letters are significantly different as determined by student’s t-test. Significantly different with compared to control (Con) by student’s t-test (*p<0.01, *p<0.001). # significantly different between AEtOH and AMEtOH by student’s t-test (#p<0.01, #p<0.001). UC: undifferentiated 3T3-L1 cell, Con: differentiated 3T3-L1 cells, AEtOH: acorn ethanol extract, AMEtOH: acorn mook ethanol extract.

Fig. 6.

Effect of AEtOH and AMEtOH on lipid accumulation in 3T3-L1 cells. The results are reported as the mean±S.D. of three independent experiment (n=3). Values with different super-script letters are significantly different as determined by student’s t-test. Significantly different with compared to control (Con) by student’s t-test (p<0.05). # significantly different between AEtOH and AMEtOH by student’s t-test (#p<0.01, #p<0.001). UC: undifferentiated 3T3-L1 cell, Con: differentiated 3T3-L1 cells without treatment with acorn ethanol extract.