Comparison of physicochemical properties of sorghum extract by ethanol concentration and its anti-adipogenic effect in 3T3-L1

DOI: https://doi.org/10.21203/rs.3.rs-1924799/v1

Abstract

Sorghum is an important cereal source of phenolic compounds, with potential health-promoting benefits. This study evaluated the phenolic content and antioxidant and anti-obesity effects of sorghum extract (SE) prepared using three solvent systems: 50% (SE50), 80% (SE80), and 100% (SE100) ethanol. The results showed that SE50 exhibited the highest total polyphenol, flavonoid, and tannin content. In addition, SE50 showed significantly higher antioxidant capacity than the other extracts, as determined using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and ferric reducing antioxidant power assays. Interestingly, the SE50 significantly inhibited lipid accumulation in 3T3-L1 preadipocytes; however, extracts using SE80 and SE100 had no such beneficial effects. Furthermore, SE50 significantly downregulated the mRNA expression of adipogenic genes (Cebpα, Pparγ, and Fabp4) and lipogenic genes (Srebp1c, Fas, and Scd1). These results suggest that the phenolic contents, antioxidant, and anti-obesity activities of 50% ethanol extract are superior to those of other extracts, and it could be used as a functional food ingredient for anti-obesity.

1. Introduction

Obesity is defined as excessive accumulation of body fat due to an imbalance in the body's caloric metabolism [1]. It poses a significant threat to public health due to being a major cause of chronic diseases, such as hypertension, type 2 diabetes, arthritis, and cancer [2]. According to reports published by the World Health Organization in 2016, 13% of adults aged 18 and over were obese, and 39% were overweight, and the prevalence of obesity is increasing in many countries. Because of this trend, reducing obesity has become an important social problem. However, anti-obesity drugs exhibit various side effects, including nausea, dizziness, and gastrointestinal disorders [3]. Therefore, recent research has focused on safe and effective anti-obesity drugs derived from natural products.

Oxidative stress can cause diseases through by excessive reactive oxygen species production and an imbalance in biological antioxidant systems [4]. Chronic oxidative stress is strongly associated with obesity. Obesity commonly causes oxidative stress, but antioxidants have not yet been proven effective in treating obesity. However, according to recent studies, polyphenols, such as resveratrol, which has antioxidant activity, may help prevent obesity [5]. In addition, some dietary phytochemicals have been documented as anti-obesity agents because they may stimulate and inhibit the differentiation of preadipocytes, thereby reducing adipose tissue mass. Taken together, natural antioxidants high in phytochemicals can promote anti-obesity effects.

Sorghum (Sorghum bicolor L.), the fifth most produced grain globally, is a rich source of nutrients and bioactive compounds [6]. Previous studies have shown that sorghum contains abundant phenolic compounds such as phenolic acid, 3-deoxy anthocyanins, flavonoids, and tannins [7]. Moreover, previous studies have reported various physiological functions of sorghum extracts, such as antioxidant activity, anti-inflammatory, antidiabetic, antibacterial, and anticancer activities [811]. In addition, a few animal studies on the anti-obesity effects of sorghum have been conducted using sorghum bran or its pigment [12, 13] However, to our knowledge, the anti-obesity effects of sorghum extract in 3T3-L1 preadipocytes have not yet been identified.

We hypothesized that sorghum extract (SE) exerts anti-obesity effects in 3T3-L1 preadipocytes. This study aimed to examine the total polyphenol, flavonoid, and tannin contents and antioxidant capacity of SE depending on solvent concentration. We further investigated how different SE influenced adipogenesis and lipid accumulation in 3T3-L1 cells by staining for lipids and measuring gene expression.

2. Materials And Methods

Materials

Sorghum (Sorghum bicolor L., Miryang 22ho) harvested in 2020 was provided by the National Institute of Crop Science (Rural Development Administration, Miryang, Korea). 3T3-L1 preadipocytes were purchased from the Korean Cell Line Bank (Seoul, Korea). Dulbecco's modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Welgene Inc. (Seoul, Korea). Fetal bovine serum (FBS) was purchased from AB Frontier Co. (Seoul, Korea). Penicillin/streptomycin (PS) and bovine calf serum (BCS) were purchased from Gibco (Carlsbad, CA, USA). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-Tirs(2-pyridyl)-s-triazine (TPTZ), 3-isobutyl-1methylxanthine (IBMX), dexamethasone (DEX), insulin, and all other analytical solvents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Sample preparation

The sorghum grains were extracted using three solvent systems: 50% (SE50), 80% (SE80), and 100% (SE100) ethanol. Sorghum was ground into a fine powder using a blender. Sorghum powder (100 g) and 50, 80, and 100% ethanol (1 L) were mixed and stirred overnight at room temperature (20 to 25°C) and filtered through filter paper (Whatman No. 1, Whatman, Maidstone, England). The filtered solution was concentrated using a rotary evaporator (Eyela, Tokyo, Japan) at 50°C. SE, obtained by lyophilization, was dissolved in DMSO to a concentration of 250 mg/mL. The samples were stored at -20°C until further analysis.

Quantification of phenolic compound

Measurement of total polyphenol content (TPC)

Quantification of TPC within SE was determined using Folin–Ciocalteu’s reagent as previously reported with some modifications [14]. Briefly, 500 µL of 10% 2 N Folin–Ciocalteu reagent was added to 200 µL of SE and allowed to react for 5 min. Then, 500 µL of 7.5% Na2CO3 was added and allowed to react at 50°C for 10 min; the absorbance was measured at 760 nm using a microplate reader (Bio Tek, Winooski, VT, USA). The TPC was expressed as mg gallic acid equivalent per 1 g SE (mg GAE/g) using a standard curve of gallic acid.

Measurement of total flavonoid content (TFC)

The TFC was estimated using an AlCl3 reagent according to a previously reported study [15]. SE (100 µL) was added to 400 µL of distilled water, and 30 µL of 5% NaNO2 and 30 µL of 10% AlCl3 were mixed. After 5 min, 200 µL of 1 M NaOH was added, and the total volume was made up to 1 mL using distilled water. Absorbance was measured at 415 nm using a microplate reader (Bio Tek). The TFC was expressed as mg quercetin equivalent per 1 g SE (mg QE/g) using a standard curve of quercetin.

Measurement of total tannin content (TTC)

The TTC was determined as previously reported with some modifications [16]. The reaction mixture was mixed with 1,000 µL of SE, 1,000 µL of 95% ethanol, and 1,000 µL of distilled water. Then, 1,000 µL of 5% Na2CO3 and 500 µL of 1 N Folin–Ciocalteu reagent were added. After incubation at room temperature for 60 min, the absorbance was measured at 725 nm using a microplate reader (Bio Tek). TTC was expressed as mg tannic acid equivalent per 1 g SE (mg TAE/g) using a standard curve of tannic acid.

Antioxidant capacity

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The radical scavenging activity of SE was assessed using DPPH free radicals, and Trolox was used as a positive control. A 20 µL sample and blank were plated with 180 µL of 0.4 mM DPPH solution in a 96-well plate. After reaction for 45 min at room temperature, the absorbance was measured at 517 nm using a microplate reader (Bio Tek). The scavenging activity of the extract was calculated using the following equation, and the data represent the IC50 value. The IC50 value is defined as the amount of antioxidants required to reduce the initial DPPH concentration by 50%.

Ac, absorbance of the control (without sample) and As, absorbance of the sample.

Ferric reducing antioxidant power (FRAP) assay

The FRAP reagent was freshly prepared for each experiment by dissolving 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3 to obtain a 10:1:1 (v/v/v) ratio. FRAP reagent was preheated at 37°C for 15 min before use. Then, 50 µL of SE at various concentrations was mixed with 150 µL of FRAP reagent and allowed to react at room temperature for 4 min. The absorbance of the mixture was measured at 595 nm using a microplate reader (Bio Tek). Reducing power was expressed as µM Trolox using a standard curve.

Cell culture

3T3-L1 preadipocytes were seeded at 3.0 × 104 cells/well in a 12-well plate and cultured in DMEM containing 10% BCS and 1% PS at 37°C and 5% CO2. When the cells were confluent (0 days), the medium was changed to differentiation medium containing DMEM, 10% FBS, IBMX (0.5 mM), DEX (1 µM), and insulin (10 µg/mL). After 2 days, the medium was replaced with fresh FBS/DMEM containing 10 µg/mL insulin for 6 days. The medium was replaced every 2 days. The cells were treated with SE (0-100 µg/mL) for 10 days to confirm the inhibitory effect on lipid accumulation. The cells were harvested after 10 days of differentiation to determine lipid accumulation.

Cytotoxicity

Cell viability was evaluated using the MTT assay. 3T3-L1 preadipocytes were cultured in a 96-well plate at 1.0 × 105 cells/well for 24 h and then treated for 24 h with 0, 25, 50, 100, and 200 µg/mL SE. Then, 20 µL of MTT solution freshly prepared at 5 mg/mL in PBS was added to each well and incubated at 37°C for a further 4 h. After incubation, the supernatant was discarded, and DMSO was added to completely solubilize the purple formazan crystals. Absorbance was measured using a 540 nm microplate reader (Bio Tek). The cell viability data were presented as percentages of the control.

Oil Red O staining

Lipid accumulation in 3T3-L1 preadipocytes was determined using Oil Red O staining. The Oil Red O stock solution was dissolved in 100% isopropanol at a concentration of 3 mg/mL and diluted with distilled water in a 3:2 volume ratio to prepare a working solution. Cells were washed twice with PBS and fixed with 10% formalin for 1 h. The cells were washed with 60% isopropanol for 5 min and stained with Oil Red O working solution for 10 min. After the cells were rinsed twice with distilled water, the stained lipid droplets were eluted with 100% isopropanol and the suspensions were centrifuged at 14,881 × g for 2 min. The absorbance of the supernatant was measured at 480 nm using a microplate reader (Bio Tek).

Real-time PCR

3T3-L1 cells were lysed to obtain total mRNA, followed by the NucleoSpin® RNA Plus isolation kit (Macherey-Nagel, Dueren, Germany). The integrity, purity, and quantity of the isolated mRNAs were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the concentrations were normalized. Subsequently, total mRNA was reverse transcribed to cDNA according to the instructions of the PrimeScript™ RT reagent kit (Takara, Shiga, Japan). Following the manufacturer’s instructions, a reaction mixture (10 µL) including 3.5 µL of mix reagent and 6 µL of total RNA were prepared. Next, mRNA expression reaction was prepared as follows: 2 µL of synthesized cDNA was mixed with 10 µL of SYBR Green Supermix, 3.2 µL of primer pair set, and 6 µL of RNase-free H2O. mRNA expression analysis was performed using a CFX96TM RT-PCR detection system (Bio-Rad, Hercules, CA, USA). The primer sequences used for real-time PCR are listed in Table 1. The relative expression levels were calculated with 36b4 as the reference gene using the delta-delta threshold cycle (ΔΔCt) method.

 
 
 
Table 1

Sequences of real-time PCR primer used in gene expression analysis.

Target

Forward primer (5ʹ-3ʹ)

Reverse primer (5ʹ-3ʹ)

36b4

TCTAGGACCCGAGAAGACCTC

GTTGTCAAACACCTGCTGGAT

Cebpα

TTACAACAGGCCAGGTTTCC

GGCTGGCGACATACAGTACA

Pparɣ

CGCTGATGCACTGCCTATGA

AGAGGTCCACAGAGCTGATTCC

Fabp4

AAGAAGTGGGAGTGGGCTTTG

CTGTCGTCTGCGGTGATTTC

Srebp1c

GAACAGACACTGGCCGAGAT

GAGGCCAGAGAAGCAGAAGAG

Fas

AGCACTGCCTTCGGTTCAGTC

AAGAGCTGTGGAGGCCACTTG

Scd1

CATCGCCTGCTCTACCCTTT

GAACTGCGCTTGGAAACCTG

Statistical analysis

Data were expressed as the mean ± standard error of the mean (SEM). All statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA, USA) using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test. Differences were considered statistically significant at p < 0.05.

3. Results And Discussion

Extraction yield, total polyphenol, total flavonoid, and total tannin content of SE.

In general, the higher the polarity of the solvent, the better physicochemical properties of the sorghum, as reported in previous studies [17]. A previous study compared ethanol and methanol extracts of sorghum grains which confirmed that the ethanol extract includes more substances such as anthocyanins, 3-deoxyanthocyanidins, flavonoids, and tannins, and improved physical and chemical properties [18]. In addition, a previous study confirmed higher phenolic contents and antioxidant capacity than methanol and acetone in ethanol extracts of sorghum leaves [19]. Generally, 50–100% (v/v) ethanol combined with water is used to extract more phenolic compounds from plant materials [20]. Therefore, we assessed three different solvents 50, 80, and 100% (v/v) ethanol.

The effects of 50, 80, and 100% ethanol on the yields, TPC, TFC, and TTC of SE are listed in Table 2. The extraction yield, affected by different ratios of ethanol/water as the extraction solvent, was the highest at SE50 (3.72%), followed by SE100 (3.57%) and SE80 (2.36%). The TPC of SE extracted using different solvent compositions was increased with decreasing ethanol concentration. The SE50 (199.00 mg GAE/g) was significantly (p < 0.05) higher than those of 144.80 mg GAE/g of the SE80 and 119.00 mg GAE/g of the SE100. These results were similar to the reported TPC of 150.08 mg GAE/g in 60% ethanol extract in sorghum grain was higher than in other ethanolic extractions [21]. As a result of TFC, the SE50 showed 321.60 mg QE/g, the SE80 showed 243.80 mg QE/g, and the SE100 showed 188.00 mg QE/g. SE50 showed significantly (p < 0.05) larger values than SE80 and SE100.

Table 2

Extraction yield, total polyphenol, total flavonoid, and total tannin contents of SE at different ethanol concentrations.

Group

Extraction yield (%)

TPC

(mg GAE/g)

TFC

(mg QE/g)

TTC

(mg TAE/g)

SE50

3.72

199.0 ± 5.7a

321.6 ± 7.0a

84.5 ± 12.4a

SE80

2.36

144.8 ± 2.8b

243.8 ± 3.2b

54.8 ± 3.8ab

SE100

3.57

119.0 ± 1.5c

188.0 ± 3.5c

44.8 ± 10.2b

Data are expressed as mean ± standard error of the mean (SEM). Means with different letters (a‒c) are significantly different (p < 0.05). SE50, 50% ethanol extract; SE80, 80% ethanol extract; SE100, 100% ethanol extract; TPC, total polyphenol content; TFC, total flavonoid content; TTC, total tannin content; GAE, gallic acid equivalent; QE, quercetin equivalent; TAE, tannic acid equivalent.

In addition, the results of the TFC showed a similar trend to that of the TPC. The results of the TTC illustrated that SE50 contained 216.20 TAE/g, while SE80 and SE100 contained 54.75 mg TAE/g and 44.82 mg TAE/g, respectively. The TPC, TFC, and TTC values increased with a decrease in ethanol concentration, a similar trend to extraction yield. These observations of the various extraction concentrations were consistent with a previous study affecting the yield and phenolic compounds [22]. Moreover, these results are comparable to previous research in which sorghum bran was effective for extracting the phenolic compounds at 50% ethanolic extraction [23]. The results could be related to the solvent polarity and the solubility of polyphenolic compounds in sorghum. Likewise, our results indicate that 50% ethanol is the best solvent for the extraction of polyphenolic compounds.

Antioxidant capacity of SE

The antioxidant capacity of SE was assessed using DPPH and FRAP assays (Table 3). The IC50 value SE50 (133.9 µg/mL) was significantly (p < 0.05) lower than SE80 (182.7 µg/mL) and SE100 (285.7 µg/mL). Since lower IC50 values indicate higher antioxidant capacity [24], radical scavenging was the highest in SE50. In contrast to a previous study, the IC50 values reported here are significantly lower [25]. As with the DPPH assay, the FRAP value of SE50 was significantly (p < 0.05) higher than SE80 and SE100. However, no significant differences were observed between the SE80 and SE100 groups. In this study, the same trend was observed as in a previous study which reported a high correlation between the phenolic compounds of the extract and DPPH radical scavenging activity [26]. Phenolic compounds are known to exhibit antioxidant properties, as phenol rings are capable of stabilizing free radicals [27]. Similarly, the phenolic compounds in sorghum are known to be responsible for its antioxidant properties [19]. The antioxidant capacity of the SE50 was higher than that of the other extracts, which could be attributed to the influence of ethanol concentration on the yield of total polyphenolics.

Table 3

The DPPH radical scavenging activity and FRAP assay of SE at different ethanol concentrations.

Group

DPPH (IC50)

FRAP (mmole TE/ 100 g)

SE50

133.9 ± 2.7a

80.17 ± 3.7a

SE80

182.7 ± 5.8b

58.10 ± 3.2bc

SE100

285.7 ± 7.8c

61.09 ± 5.2c

Data are expressed as mean ± standard error of the mean (SEM). Means with different letters (a‒c) are significantly different (p < 0.05). SE50, 50% ethanol extract; SE80, 80% ethanol extract; SE100, 100% ethanol extract; TE, Trolox equivalents; DPPH, 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay; FRAP, ferric ion reducing antioxidant power assay.

Cytotoxicity of SE in 3T3-L1 preadipocytes

The cytotoxicity of SE was assessed using the MTT assay in 3T3-L1 preadipocytes (Fig. 1). Treatment of 3T3-L1 preadipocytes with 0, 25, 50, 100, and 200 µg/mL concentrations of each ethanol extract for 24h revealed no cytotoxic effect, with cell viability remaining high at > 80% after treatment. However, the viability of cells treated with SE80 decreased by approximately 17 and 19% at concentrations above 100 µg/mL. In addition, the concentration of 200 µg/mL was significantly reduced by approximately 19% compared with control. These results showed that the SE80 indicated cytotoxicity against 3T3-L1 cells at 200 µg/mL. On the other hand, cell viability SE treatment up to 200 µg/mL in SE50 and SE100 that indicated 15% or less had no cytotoxicity. Thus, we performed SE concentrations for subsequent experiments at 25 to 100 µg/mL.

Effect of SE on lipid accumulation in 3T3-L1 preadipocytes

The potential lipid-inhibiting effect of SE was investigated using Oil Red O staining of 3T3-L1 differentiated adipocytes. The effects of lipid accumulation using a microscope and quantified intracellular lipid contents at different ethanol concentrations in SE at 0, 25, 50, and 100 µg/mL are illustrated in Fig. 2. SE50, SE80, and SE100 at 25 µg/mL were significantly reduced by 87, 90, and 86%, respectively, compared to the control group (Fig. 2a). However, SE80 respectively decreased by 91% and 89% at concentrations of 50 and 100 µg/mL, and there was no significant difference. On the other hand, SE100 decreased by 85% and 86%, and the SE50 showed significant differences up to 79% and 75% (Figs. 2b and c). In summary, SE50 showed a greater inhibition ability of lipid accumulation than SE80 and SE100. Based on these findings, SE50 was selected for subsequent examination of the molecular mechanism.

Effect of sorghum SE50 on the mRNA expression levels of Cebpα, Pparγ, and Fabp4 in 3T3-L1 preadipocytes

Real-time RT-PCR analysis was conducted to determine whether adipogenesis affects the expression of a transcription factor. The mechanism of inhibition of lipid accumulation by the SE50 was investigated by analyzing the mRNA expression of genes that modulate adipogenic CCATT/enhancer-binding protein-α (Cebpα), peroxisome proliferator-activated receptor (Pparγ), and fatty acid-binding protein (Fabp4).

The most critical first transcription factors for adipogenesis are the expression of Cebpα and the nuclear hormone receptor Pparγ; these two factors remain elevated for adipocyte maturation. Pparγ induces the expression of the adipogenic transcription factor Cebpα and then binds with Cebpα to the promoter/enhancer of the gene encoding the adipocyte fatty acid-binding protein Fabp4 [28]. Fabp4, expressed at the last stage of adipocyte differentiation, is involved in fatty acid synthesis, transport, storage, and energy consumption [29]. As shown in Fig. 3, SE50 induced significant down-regulation of gene expression of Cebpα, Pparγ, and Fabp4 (p < 0.05). The relation level of Cebpα, Pparγ, and Fabp4 mRNA expression decreased by 65, 33, and 47%, respectively, following treatment with SE50 at a concentration of 100 µg/mL. Similarly, sorghum bran extract repressed the expression of adipogenic genes Cebpα and Pparγ [30]. Therefore, SE50 inhibits the differentiation of adipocytes by mutually regulating the expression of these genes.

Effect of SE50 on the mRNA expression levels of Srebp1c, Fas, and Scd1 in 3T3-L1 preadipocytes

The mechanism of lipogenesis inhibition affects the mRNA expression levels of lipid metabolism-related genes of sterol regulatory element binding protein 1 (Srebp1c), fatty acid synthase (Fas), and stearoyl-CoA desaturase 1 (Scd1). SREBP-1 induces intracellular triglyceride production and accumulation by increasing the expression of lipogenic enzymes, such as Scd1 and Fas, which promote preadipocyte differentiation and increase the content of lipid droplets in mature adipocytes [31]. The results of treatment with SE50 are shown in Fig. 4. The gene expression of Srebp1c was significantly (p < 0.05) reduced by 19% and 27% at concentrations of 50 and 100 µg/mL, respectively, compared to the control. Fas and Scd1 expression were also significantly (p < 0.05) decreased by 19% and 18%, respectively, at 100 µg/mL. These results were consistent with the results of previous studies showing that the reduction of Srebp1c expression in 3T3-L1 preadipocytes suppressed the mRNA and protein expression of adipogenic enzymes such as Fas and Scd1 [32]. As a result, SE50 downregulated the expression of Cebpα, Pparγ, and Fabp4 in 3T3-L1 preadipocytes to suppress the early stage of adipogenesis of adipogenic and adipogenic enzymes genes such as Srebp1c, Fas, and Scd1. It was confirmed that effectively inhibited fatty acid metabolism and lipid accumulation.

Conclusions

Our results showed that sorghum has many phenolic compounds and antioxidants, as evidenced in numerous previous studies. When the effects of different ethanol concentrations were confirmed, phenolic compounds and the antioxidant capacity were the highest in SE50 compared to SE80 and SE100. We confirmed that ethanol concentration could influence phenolic compounds and antioxidant capacity. We also demonstrated that SE50 exerted a significant anti-obesity effect by decreasing adipogenic and lipogenic transcription factors by suppressing the mRNA expression of Cebpα, Pparγ, Fabp4, Srebp1c, Fas, and Scd1 in 3T3-L1 preadipocytes. In conclusion, these findings provide convincing evidence to indicate that SE extracted using 50% ethanol could be used as a bioactive ingredient in functional foods for anti-obesity. Further studies are needed to identify specific phenolic compounds with anti-obesity effects.

Declarations

Author contributions

Seyoung Jung: Conceptualization, Methodology, Validation, Investigation, Data Curation, Writing - Original Draft, Visualization. Eun Woo Jeong: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - Review & Editing. Youjin Baek: Validation, Writing - Review & Editing. Gwang-woong Go*: Conceptualization, Methodology, Writing - Review & Editing. Hyeon Gyu Lee*: Conceptualization, Methodology, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. All authors read and approved the final manuscript.

Funding

This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of miscellaneous cereal varieties for mechanization and cropping system, Project No. PJ015056012022)" Rural Development Administration, Republic of Korea.

Data Availability

The data presented in this study are available within the article.

Ethics Approval

Not applicable

Consent to Participate

Not applicable

Consent for Publication

Not applicable

Conflict of Interest

The authors have no conflicts of interest.

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