Obesity is primarily caused by excessive lipid accumulation because of an imbalance between energy expenditure and intake (Hwang et al., 2015; Tsatsoulis and Paschou, 2020). CCAAT/enhancer-binding protein-alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), and adipocyte protein 2 (aP2) (Ramji and Foka, 2002; Dunning et al., 2014; Floresta et al., 2017) are important transcription factors involved in promoting adipogenesis. Among them, PPARγ acts as a fatty acid sensor, regulating genes associated with lipid metabolism and exclusively inducing aP2 expression in adipocytes and macrophages (Tontonoz et al., 1994; Kuryłowicz, 2020). aP2 functions as a fatty acid chaperone, linking intracellular lipids to biological targets and signaling pathways (Boord et al., 2002.; Liu et al., 2022). Therefore, inhibiting the key transcription factors involved in adipocyte differentiation may be used to control obesity (Guru et al., 2021). According to recent studies, soy isoflavones can regulate the expression of PPARγ, C/EBPα, and aP2 (Choi et al., 2020).

Soybeans contain various bioactive compounds, including phenolic acids, saponins, trypsin inhibitors, peptides, and isoflavones. Among them, isoflavones, a group of flavonoids derived from phenolic compounds, can influence chronic diseases, including cardiovascular disease, immune disorders, insulin-resistance-related diabetes, and obesity (Chatterjee et al., 2018; Hsiao et al., 2020). Isoflavones are predominantly found in soybeans as nonhydrophilic glucosides attached to malonyl and acetyl groups (Kim et al., 2010). According to Izumi et al. (2000), isoflavone aglycones (daidzein and genistein) are more bioavailable and effective in maintaining high isoflavone concentrations in human plasma compared with their glycosidic forms (daidzin and genistin). Daidzein and genistein are secondary metabolites with significant effects on human health, including inhibiting the proliferation of 3T3-L1 adipocytes (Kim et al., 2010; He et al., 2016; Hsiao and Hsieh, 2018). Processing techniques, including thermal treatment, fermentation, and enzymatic hydrolysis, can influence the content, composition, and profile of isoflavones in soy-based foods (Wang and Murphy, 1994; Huang et al., 2014).

Bionuruk^TM (a traditional Korean fermentation starter made by fermenting wheat bran with Rhizopus oryzae and used to saccharify rice) contains β-glucosidase enzymes. These enzymes play an important role in hydrolyzing isoflavone glucosides into their aglycone forms (Mei et al., 2019; Delgado et al., 2021). In the present study, we hypothesized that reacting soybeans with Bionuruk^TM would effectively convert isoflavone glycosides into their corresponding aglycones. Furthermore, we expected that increasing the Bionuruk^TM concentration would result in a higher conversion rate within the same time frame. Therefore, this study aimed to investigate the conversion of soy isoflavone glycosides to their aglycone forms by reacting soybeans with Bionuruk^TM and to evaluate the effects of these conversions on lipid accumulation and the mRNA and protein expression levels of adipogenesis-related transcription factors in 3T3-L1 adipocytes.

Materials

Soybeans (Glycine max) were harvested from a domestic agricultural farm in Miryang, South Korea. All solvents [high-performance liquid chromatography (HPLC)-grade] were purchased from Daejeong Chemical Co. Bionuruk^TM was purchased from Korea Enzyme Inc. The reference standard materials for isoflavone glucosides (daidzin, glycitin, and genistin) and isoflavone aglycones (daidzein, glycitein, and genistein) were purchased from Sigma-Aldrich. 6’-Malonyl-glucoside (6’-O-malonyldaidzin, 6’-O-malonyldaidzin) was purchased from Nagara Science Co. 3T3-L1 preadipocytes were obtained from the Korean Cell Line Bank. Oil Red O, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and insulin were purchased from Sigma-Aldrich. The Total RNA Extraction Kit (MK14115) and 2X One-Step RT-PCR (reverse transcription polymerase chain reaction) Master Mix SYBR Green (MR03011) were purchased from CancerROP Co., Ltd. Dulbecco’s Modified Eagle Medium (DMEM), bovine calf serum (BCS), and fetal bovine serum (FBS) were purchased from Welgene, Inc.

Preparation of soybean extracts

The extract from nonreacted soybeans (SE-N) was obtained by extracting raw ground soybeans with 80% ethanol. The soybean extract prepared with Bionuruk^TM (SE-B) was obtained by reacting the soybean powder with different Bionuruk^TM concentrations (1%, 5%, 10%, and 50%), followed by extraction with 80% ethanol. The resulting extracts were designated as SE-B1, SE-B5, SE-B10, and SE-B50, respectively.

Isoflavone profile analysis

HPLC analysis of isoflavones was performed in accordance with the method of da Silva et al. (2011). The analysis was conducted using the ACME 9000 HPLC System (Youngin Chromass Co.), comprising the Autochro-3000 software, an ACME 9000 pump, an ACME 9000 column oven, and an ultraviolet detector. The Shim-pack GIS C18 Column (250 mm×4.6 mm, 5 µm) (Shimadzu Co.) was used, with the oven temperature set to 30°C. Chromatograms were recorded at 254 nm for quantitative analysis. The samples were eluted at a flow rate of 0.5 mL/min using a linear gradient of 5.0% (v/v) acetic acid in water (A) and methanol (B). The conversion of isoflavone glucoside content to isoflavone aglycone content was calculated by multiplying by the conversion coefficient (1/1.6), as indicated by Lee and Chung (2022).

Culture and differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes were cultured at 37°C in 5% CO₂. The growth medium comprised DMEM (89%) supplemented with BCS (10%) and penicillin/streptomycin (1%). 3T3-L1 preadipocytes (5×10⁴ cells/mL) were seeded in 12-well plates for differentiation and grown to 100% confluency over five days. Following treatment with SE-B samples (SE-B1, SE-B5, and SE-B10) at 1.35, 2.7, and 5.4 µg/mL, respectively, confluent 3T3-L1 preadipocytes were induced to differentiate by adding 1 µM DEX and 0.5 mM IBMX in DMEM with 10% FBS. Two days after the treatment, the medium was replaced with fresh medium containing 10% FBS and 10 µg/mL of insulin, and this was repeated every two days for eight days.

Assessment of 3T3-L1 preadipocyte viability

The cell viability of 3T3-L1 preadipocytes treated with SE-B samples was measured using an MTT assay in accordance with the method of Huang et al. (2014). 3T3-L1 preadipocytes were seeded at a density of 1×10⁴ cells/mL in 96-well plates and cultured for 24 h. The SE-B samples (SE-B1, SE-B5, and SE-B10) were diluted to concentrations of 0, 1.35, 2.7, and 5.4 µg/mL and added to the wells. The control group comprised 50 mM potassium phosphate buffer (pH 6.8), and the blank group contained the growth medium of 3T3-L1 preadipocytes. After 24 h of incubation, 20 µL of 5 mg/mL MTT solution was added, and the cells were further incubated for 2 h in the dark. After adding 200 µL of dimethyl sulfoxide, the absorbance was measured at 570 nm, and the cell viability was calculated as follows:

Cell viability (%)=[(Abs_sample−Abs_blank)/(Abs_control−Abs_blank)]×100

Lipid accumulation

To determine lipid accumulation, Oil Red O staining was performed in accordance with the method of Choi et al. (2020) with modifications. After differentiation, 3T3-L1 adipocytes were fixed with 10% formalin for 1 h and stained with filtered Oil Red O solution for 10 min. Subsequently, the cells were washed with distilled water and imaged using the Olympus CKX53 Microscope. The Oil Red O in stained cells was eluted with 100% isopropanol, and the absorbance was measured at 492 nm using a microplate reader. The results are expressed as a percentage of control cells (fully differentiated cells without sample treatment) using the following equation:

Oil red O quantification (%)=[(Abs_sample−Abs_blank)/(Abs_control−Abs_blank)]×100

mRNA expression

To assess the mRNA expression of adipogenesis-related transcription factors (PPARγ, C/EBPα, and aP2) in differentiated 3T3-L1 adipocytes, total RNA was isolated using the Total RNA Extraction Kit (MK14115, MGmed). The RNA concentration was measured using the Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments Inc.) and quantified at 50 ng/µL. Quantitative RT-PCR was performed using the QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific Inc.). Reverse transcription was performed at 42°C for 30 min, followed by denaturation at 95°C for 30 s, annealing at 55°C for 10 s, and elongation at 72°C for 30 s for 40 cycles.

The following primer sequences were used for quantitative RT-PCR: for PPARγ, the forward primer is 5’-AGGCTTCCACTATGGAGTTC-3’, and the reverse primer is 5’-CCAACAGCTTCTCCTTCTC-3’. For C/EBPα, the forward primer is 5’-CAAGAACAGCAACGAGTACC-3’, and the reverse primer is 5’-TTGACCAAGGAGCTCTCA-3’. For aP2, the forward primer is 5’-GGATTTG GTCACCATCCGGT-3’, and the reverse primer is 5’-TTCACCTTCCTGTCGTCTGC-3’. For glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the forward primer is 5’-AACTTTGGCATTGTGGAAGG-3’, and the reverse primer is 5’-ACACATTGGGGGTAGGAACA-3’. The expression of the target genes was normalized to GAPDH, which served as the control gene. The relative mRNA expression levels were calculated using the comparative cycle threshold (2^−ΔΔCT) method. The primer sequences were taken from Choi et al. (2020) and Moon et al. (2013).

Protein expression

The proteins that were extracted from each sample were used for Western blot analysis. The protein concentrations were determined using the Bradford assay. The proteins (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% to 15% acrylamide gel and transferred to a polyvinylidene fluoride membrane. The membranes were incubated with the primary antibodies of PPARγ, C/EBPα, and aP2 and GAPDH (1:200 dilution in 3% bovine serum albumin) at 4°C for 24 h, followed by incubation with horseradish peroxidase-conjugated anti-mouse immunoglobulin secondary antibodies (1:2,000 dilution in 5% skim milk) at room temperature for 2 h. The membranes were washed thrice with Tris Buffered Saline with 0.05% Tween^Ⓡ 20, developed using an enhanced chemiluminescence solution (Dynebio Inc.), and imaged with the ImageQuant LAS 500 system (Cytiva Inc.). The relative protein expression was quantified using ImageJ software.

Statistical analysis

Data are presented as the mean±standard deviation of triplicate values. The Statistical Package for the Social Sciences (SPSS) software (Version 21.0, SPSS Inc.) was used to calculate statistical significance using Duncan’s multiple range test and one-way analysis of variance (ANOVA). Statistical significance was considered at P<0.05.

Isoflavone profiles

Calibration curves were prepared using standard isoflavone compounds to analyze the isoflavone content of SE-B samples. The calibration curves exhibited high correlation coefficients (R²=0.99) within the selected concentration range. Fig. 1 shows the chromatograms of SE-B samples, which were compared with the retention times of the standard isoflavone compounds. Table 1 presents the analytical results for isoflavone glycosides and aglycones in SE-B samples. The total isoflavone content is depicted as a combination of glycosides (malonyldaidzin, malonylgenistin, genistin, glycitin, and daidzin) and aglycones (genistein, glycitein, and daidzein). The amount of isoflavone glycosides was converted into the corresponding amount of isoflavone aglycones using a conversion coefficient of 1/1.6, as provided by Lee and Chung (2022).

Figure 1. HPLC chromatograms of soybean extracts prepared with Bionuruk^TM. (A) Soybean extracts prepared without Bionuruk^TM, (B) soybean extracts prepared with Bionuruk^TM (1%), (C) soybean extracts prepared with Bionuruk^TM (5%), (D) soybean extracts prepared with Bionuruk^TM (10%), and (E) soybean extracts prepared with Bionuruk^TM (50%). a, daidzin; b, glycitin; c, genistin; d, malonyldaidzin; e, malonylgenistin; f, daidzein; g, glycitein; h, genistein.

Table 1 . Isoflavone content in soybean extracts (SE-B) prepared with Bionuruk^TM.

Isoflavones (µg/g)	SE-N1)	SE-B12)	SE-B53)	SE-B104)	SE-B505)
Malonyldaidzin	30.5±1.5a	2.5±0.3b	n.d	n.d	n.d
Malonylgenistin	59.6±7.1a	6.5±0.2bc	2.9±0.3cd	0.1±0.0e	n.d
Sum of malonylglucosides	90.1±5.8a	9.1±0.1b	2.9±0.3c	0.1±0.0d	n.d
Daidzin	36.4±0.6a	4.8±0.2b	3.7±0.2c	3.4±0.1c	2.8±0.4d
Glycitin	14.2±0.3a	1.3±0.3b	n.d	n.d	n.d
Genistin	69.6±1.2a	7.4±0.2b	2.5±0.1c	1.5±0.1d	2.6±0.3c
Sum of glycosides	120.3±0.9a	13.5±0.2b	6.2±0.1c	4.9±0.1d	5.4±0.3d
Daidzein	25.6±0.5e	217.2±3.7c	226.6±7.2b	236.3±3.8a	161.0±4.0b
Glycitein	5.8±0.9d	69.4±0.2c	71.4±0.2b	73.1±0.3a	69.8±1.3c
Genistein	26.6±1.1e	299.1±3.1c	329.1±15.4b	345.8±4.4a	237.7±5.5d
Sum of aglycones	58.0±0.4e	585.6±0.8c	627.1±11.1b	655.2±1.8a	468.4±3.3d
Total isoflavones (aglycones)	189.5±2.7e	599.6±0.8c	632.8±11.5b	658.4±1.8a	471.8±3.3d

Values are presented as mean±SD of triplicate values..

¹⁾Soybean extracts prepared without Bionuruk^TM, ²⁾soybean extracts prepared with Bionuruk^TM (1%), ³⁾soybean extracts prepared with Bionuruk^TM (5%), ⁴⁾soybean extracts prepared with Bionuruk^TM (10%), and ⁵⁾soybean extracts prepared with Bionuruk^TM (50%)..

Means in the same row that do not share a common letter (a-e) are significantly different (P<0.05) according to Duncan’s multiple range test..

n.d, not detected..

The total isoflavone content in SE-N was 189.5 µg/g, and 69.4% were found in the form of malonylglucosides and glycosides. The glycoside distribution was as follows: malonyldaidzin (10.1%), malonylgenistin (19.7%), daidzin (12%), glycitin (4.7%), and genistin (23%). Meanwhile, the aglycone content of SE-N was 30.6%, with daidzein (13.5%), glycitein (3.1%), and genistein (14%) accounting for the remainder.

The SE-B samples (SE-B1, SE-B5, SE-B10, and SE-B50) exhibited a significant increase in the total isoflavone content, with 3.2, 3.3, 3.5, and 2.5 times higher values than SE-N, respectively. In addition, the levels of isoflavone malonylglucosides were lower in SE-B samples than in SE-N. By contrast, the levels of isoflavone aglycones were higher in SE-B samples than in SE-N. In SE-B1, SE-B5, SE-B10, and SE-B50, the percentage of isoflavone glycosides decreased to 2.3%, 0.9%, 0.5%, and 0.7% of the total isoflavone content, whereas that of isoflavone aglycones increased to 97.6%, 99.1%, 99.5%, and 99.3%, respectively. For example, in SE-B10, the malonylglucoside content decreased from 90.1 µg/g to almost undetectable levels after reacting with Bionuruk^TM, whereas the isoflavone aglycone content increased from 58.0 µg/g to 655.2 µg/g. These results indicate that isoflavone glycosides were successfully converted to isoflavone aglycones.

Effects of standard isoflavones and SE-B on the viability of 3T3-L1 preadipocytes

The viability of 3T3-L1 preadipocytes was assessed after treatment with standard isoflavones and SE-B samples (Fig. 2). The preadipocytes were treated with vehicle, SE-B1, SE-B5, SE-B10, genistin, and genistein at concentrations of 1.35, 2.7, and 5.4 µg/mL. No significant differences in cell viability were observed at these concentrations. At the highest concentration (5.4 µg/mL), the viability of 3T3-L1 preadipocytes treated with SE-B1, SE-B5, SE-B10, genistin, and genistein was 101%, 103%, 102%, 105.7%, and 105.8%, respectively. A cell viability greater than 90% indicates no cytotoxicity (Li et al., 2008). Based on this criterion, none of the treatments caused toxicity toward the preadipocytes. These results are consistent with those reported by Choi et al. (2020), who found that genistein is a potent antiadipogenic agent. After confirming the cell viability, we proceeded with subsequent assays.

Figure 2. Viability of 3T3-L1 preadipocytes using standard isoflavones and soybean extracts prepared with Bionuruk^TM. Differences between data were analyzed using one-way ANOVA with Duncan’s multiple range test. Values are presented as mean±SD of triplicate values. Vehicle, 50 mM potassium phosphate buffer; SE-B1, soybean extracts prepared with Bionuruk^TM (1%); SE-B5, soybean extracts prepared with Bionuruk^TM (5%); SE-B10, soybean extracts prepared with Bionuruk^TM (10%).

Effects of standard isoflavones and SE-B on lipid accumulation in 3T3-L1 adipocytes

The effects of standard isoflavones and SE-B on the differentiation of 3T3-L1 preadipocytes were observed by microscopy after Oil Red O staining (Fig. 3A and 3B). After staining, Oil Red O was destained with isopropanol, and lipid accumulation was quantified using a microplate reader (Fig. 3C and 3D). Genistein treatment resulted in a more significant reduction in the number of lipid droplets compared with genistin treatment (Fig. 3A and 3C). At 1.35, 2.7, and 5.4 µg/mL, genistin treatment resulted in lipid accumulation of 83.2%, 81.2%, and 77.1%, respectively, whereas genistein treatment resulted in lipid accumulation of 68.8%, 66.3%, and 56.6%, respectively.

Figure 3. Effects of standard isoflavones and soybean extracts prepared with Bionuruk^TM on lipid accumulation in 3T3-L1 adipocytes. (A) Oil Red O staining image of 3T3-L1 adipocytes treated with standard isoflavones and (B) SE-B. (C) Lipid accumulation in 3T3-L1 adipocytes treated with standard isoflavones and (D) SE-B. Bars with different letters (a-d) indicate significant differences (P<0.05) according to Duncan’s multiple range test. Values are presented as mean±SD of triplicate values. Vehicle, 50 mM potassium phosphate buffer; SE-B1, soybean extracts prepared with Bionuruk^TM (1%); SE-B5, soybean extracts prepared with Bionuruk^TM (5%); SE-B10, soybean extracts prepared with Bionuruk^TM (10%).

SE-B10 treatment exhibited a more pronounced decrease in the lipid droplet number compared with SE-B1 and SE-B5 treatments (Fig. 3B and 3D). At 1.35, 2.7, and 5.4 µg/mL, SE-B1 treatment resulted in lipid accumulation of 82.9%, 74.1%, and 69.5%, respectively; SE-B5 treatment resulted in lipid accumulation of 79.0%, 69.7%, and 61.7%, respectively; and SE-B10 treatment resulted in lipid accumulation of 68.9%, 62.6%, and 50.3%, respectively.

Effects of SE-B on the mRNA expression of 3T3-L1 adipocytes

The inhibition of lipid accumulation was further examined in relation to the mRNA expression of key transcription factors (PPARγ, C/EBPα, and aP2) (Fig. 4). The mRNA expression levels of PPARγ, C/EBPα, and aP2 typically increase during preadipocyte differentiation. However, the mRNA expression levels were significantly reduced in 3T3-L1 adipocytes treated with SE-B samples (SE-B1, SE-B5, and SE-B10). Notably, the mRNA expression of these genes was most suppressed in the SE-B10 treatment group, suggesting a more pronounced inhibition of adipogenesis. The mRNA expression levels of PPARγ, C/EBPα, and aP2 were downregulated in a dose-dependent manner. At 5.4 µg/mL, the relative mRNA expression levels of PPARγ were 66.7% for SE-B1, 59.9% for SE-B5, and 54.2% for SE-B10; those of C/EBPα were 63.9% for SE-B1, 55.3% for SE-B5, and 49.3% for SE-B10; and those of aP2 were 60.3% for SE-B1, 52.9% for SE-B5, and 43.1% for SE-B10.

Figure 4. Effects of soybean extracts prepared with Bionuruk^TM on the mRNA expression of 3T3-L1 adipocytes. mRNA expression of (A) PPARγ, (B) C/EBPα, and (C) aP2. Bars with different letters (a-d) indicate significant differences (P<0.05) according to Duncan’s multiple range test. Values are presented as mean±SD of triplicate values. Vehicle, 50 mM potassium phosphate buffer; SE-B1, soybean extracts prepared with Bionuruk^TM (1%); SE-B5, soybean extracts prepared with Bionuruk^TM (5%); SE-B10, soybean extracts prepared with Bionuruk^TM (10%); PPARγ, peroxisome proliferator-activated receptor gamma; C/EBPα, CCAAT/enhancer-binding protein-alpha; aP2, adipocyte protein 2.

Effects of SE-B on protein expression in 3T3-L1 adipocytes

Western blot analysis was performed to confirm the effects of SE-B on protein expression, particularly for PPARγ, C/EBPα, and aP2 (Fig. 5). The protein expression levels of these transcription factors were significantly lower in the SE-B-treated groups than in the control group. SE-B10 treatment significantly downregulated PPARγ, C/EBPα, and aP2 expression. In particular, the protein expression levels were most reduced at a concentration of 5.4 µg/mL: PPARγ (67.9% for SE-B1, 58.9% for SE-B5, and 51.3% for SE-B10), C/EBPα (75.9% for SE-B1, 66.6% for SE-B5, and 58.4% for SE-B10), and aP2 (80.2% for SE-B1, 75.4% for SE-B5, and 71.7% for SE-B10). The protein expression levels were inhibited in a dose-dependent manner.

Figure 5. Effects of soybean extracts prepared with Bionuruk^TM on the protein expression of 3T3-L1 adipocytes. Protein expression of (A) PPARγ, (B) C/EBPα, (C) aP2, and (D) Western blot images. Bars with different letters (a-d) indicate significant differences (P<0.05) according to Duncan’s multiple range test. Values are presented as mean±SD of triplicate values. Vehicle, 50 mM potassium phosphate buffer; SE-B1, soybean extracts prepared with Bionuruk^TM (1%); SE-B5, soybean extracts prepared with Bionuruk^TM (5%); SE-B10, soybean extracts prepared with Bionuruk^TM (10%); PPARγ, peroxisome proliferator-activated receptor gamma; C/EBPα, CCAAT/enhancer-binding protein-alpha; aP2, adipocyte protein 2.

Soybean isoflavones exist in four primary chemical forms: aglycones, glucosides, acetylglucosides, and malonylglucosides (Zubik and Meydani, 2003). They are predominantly found as nonhydrophilic β-glycosides attached to malonyl and acetyl groups; in nature, flavonoids almost exclusively exist as β-glycosides (Ioku et al., 1998; Kim et al., 2021). In the small intestine and liver, isoflavone glucosides are hydrolyzed to their aglycone forms by β-glucosidases (Day et al., 1998). For example, the enzyme β-glucosidase produced by Rhizopus spp. converts isoflavone glycosides in black soybeans to aglycones (Cheng et al., 2010). In the present study, the use of Bionuruk^TM increased the isoflavone aglycone content in SE-B from 58.0 µg/g to 655.2 µg/g. This is similar to the results of Kim et al. (2021), who used a roasting process, and Lee et al. (2014), who used Bacillus strains to convert isoflavone glycosides to aglycones. However, the present study did not involve high temperatures or extended fermentation. In addition, the increase in the total isoflavone content from 189.5±2.7 to 658.4±1.8 is consistent with the results of da Silva et al. (2011), who used fermentation with soybean flour.

PPARγ, C/EBPα, and aP2 are critical transcription factors that regulate adipogenesis. PPARγ promotes adipocyte differentiation, whereas C/EBPα induces PPARγ expression and contributes to adipocyte maturation. aP2 is involved in fat metabolism, regulating the transport and storage of fatty acids in adipocytes (Rosen et al., 1999; Ramji and Foka, 2002; Kahn et al., 2006; Floresta et al., 2017). The downregulation of these transcription factors inhibits adipocyte differentiation and reduces fat accumulation, as observed in the present study. Isoflavones, particularly genistein, have been found to inhibit adipocyte differentiation and lipid accumulation in 3T3-L1 adipocytes (Yanagisawa et al., 2012; Gao et al., 2015; Choi et al., 2020). The findings of the present study suggest that the conversion of isoflavone glycosides to aglycones using Bionuruk^TM enhances the antiadipogenic effects, as evidenced by a significant reduction in lipid accumulation and the downregulation of PPARγ, C/EBPα, and aP2 expression. In a comparison between genistin and genistein, the aglycone form of isoflavones was found to be more effective in inhibiting lipid accumulation than the glycoside form. This result was similar to the findings of Choi et al. (2007). Therefore, SE-B, which had a higher isoflavone aglycone content, greatly inhibited lipid accumulation. Among the SE-B samples, SE-B10, which contained the highest aglycone content, showed the strongest inhibitory effect on lipid accumulation. Moreover, the effects were dose-dependent. The inhibition of lipid accumulation was associated with a decrease in the mRNA expression of PPARγ, C/EBPα, and aP2, which influence adipogenesis. The mRNA expression levels of PPARγ, C/EBPα, and aP2 in SE-B were lower than those in SE-N, indicating that isoflavone aglycones affected the reduction in mRNA expression of these genes, with these effects being dose-dependent. In addition, the protein expression levels of PPARγ, C/EBPα, and aP2 in SE-B were lower than those in SE-N, and these results were dose-dependent. These findings were similar to those of Gao et al. (2015), who reported a decrease in the mRNA expression of PPARγ and C/EBPα and the protein expression of aP2 in 3T3-L1 adipocytes treated with isoflavones from chickpeas. In conclusion, the conversion of soybean isoflavone glycosides to aglycones using Bionuruk^TM significantly enhances the antiadipogenic effects of soybean extracts, potentially offering a novel approach to regulate adipogenesis and inhibit fat accumulation in human adipocytes.

None.

The authors declare no conflict of interest.

Concept and design: HC. Analysis and interpretation: HC, HK. Data collection: HC, HK. Writing the article: HC, HK. Critical revision of the article: HC, HK. Final approval of the article: all authors. Statistical analysis: HC, HK. Overall responsibility: HC.