Inhibitory E ﬀ ects of Thymol Isolated from Curcuma longa L. on Adipogenesis in HepG2 Cells

: Non-alcoholic fatty liver disease (NAFLD) is a disease associated with metabolic syndromes such as diabetes and obesity, regardless of alcohol consumption, and refers to the accumulation of triacylglycerols in the liver. Thymol (THY) is a vegetable essential oil that is naturally contained in the Zingiberaceae and Lamiaceae families. THY was isolated from Curcuma longa L. The rhizomes of Curcuma longa L. were dried, sliced and extracted with 50% ethanol and then isolated through repeated column chromatography. This study was conducted to investigate the inhibitory e ﬀ ect of THY, even in non-alcoholic fatty liver disease, in relation to the inhibiting hyperlipidemia e ﬀ ect of THY, which was demonstrated in previous studies. Hepatocytes were treated with oleate (OA) containing THY to observe lipid accumulation by Oil Red O staining (ORO). We also tested the e ﬀ ect of THY on triacylglycerols (TG) and total cholesterol (TC) in HepG2 cells. Western blot and real-time RT-PCR using sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), CCAAT-enhancer-binding protein (C / EBP), proliferator-activated receptor γ (PPAR γ ), and adenosine monophosphate (AMP)-activated protein kinase (AMPK) expressions were carried out. Consequently, inhibition of lipogenesis by THY (100 µ M or 200 µ M) in NAFLD treated with OA in HepG2 cells was conﬁrmed. The results of TG and TC experiments conﬁrmed a decrease in the degree of fat accumulation in the liver. Furthermore, inhibition of the SREBP-1c, FAS, ACC, C / EBP and PPAR γ expressions that mediated fat accumulation and increased AMPK phosphorylation was observed. Taken together, THY is proposed as a potential natural constituent for the treatment of NAFLD. THY 100 (100 μM), and THY 200 (200 μM) reduced the expression of genes involved in lipid production (SREBP-1c, ACC, FAS, C/EBP, and PPARγ). These results suggest that SM (20 μg), THY 100 (100 μM), and THY 200 (200 μM) reduce hepatic lipid accumulation.


Introduction
Fatty liver disease is caused by the buildup of excess fat in the liver. In general, fatty liver disease is defined as the accumulation of triacylglycerols in more than 5% of hepatocytes [1]. Fatty liver disease can be divided into two categories: alcoholic and non-alcoholic. Alcoholic fatty liver disease occurs under the condition of heavy alcohol drinking, as the name reveals. In contrast, fatty liver disease associated with diabetes, obesity, and hyperlipidemia is generalized under the term non-alcoholic fatty liver disease (NAFLD), regardless of alcohol consumption [2]. NAFLD appears in relation to metabolic syndrome [3]. NAFLD tends to impact not only simple fatty liver disease, but also chronic hepatitis

HepG2 Cells' Viability Based on Thymol (THY)
The MTS assay was performed to carry out cytotoxicity tests in the presence of a varying concentration of oleate (OA), CE, silymarin (SM), and THY. As shown in the Figure 2, toxicity could not be confirmed in the presence of CE (200 μg), SM (20 μg), or THY (50, 100, 200, and 400 μM). In particular, an increase in cellular activity was observed when the cells were treated with drugs after OA (500 μM) treatment.

HepG2 Cells' Viability Based on Thymol (THY)
The MTS assay was performed to carry out cytotoxicity tests in the presence of a varying concentration of oleate (OA), CE, silymarin (SM), and THY. As shown in the Figure 2, toxicity could not be confirmed in the presence of CE (200 µg), SM (20 µg), or THY (50, 100, 200, and 400 µM). In particular, an increase in cellular activity was observed when the cells were treated with drugs after OA (500 µM) treatment.

HepG2 Cells' Viability Based on Thymol (THY)
The MTS assay was performed to carry out cytotoxicity tests in the presence of a varying concentration of oleate (OA), CE, silymarin (SM), and THY. As shown in the Figure 2, toxicity could not be confirmed in the presence of CE (200 μg), SM (20 μg), or THY (50, 100, 200, and 400 μM). In particular, an increase in cellular activity was observed when the cells were treated with drugs after OA (500 μM) treatment.

Effects on Intracellular Lipid Accumulation in HepG2 Cells
Before investigating the inhibitory effect of fat accumulation by THY, the degree of fat pinching was confirmed through Oil Red O staining in CE and tested with THY (CE data not shown). Fat accumulation was induced normally by intracellular OA (500 μM) treatment alone. As shown in Figure 3, suppression of fat accumulation was observed in the THY treatment group and, in particular, THY showed a significant fat suppression effect at 200 μM.

Effects on Intracellular Lipid Accumulation in HepG2 Cells
Before investigating the inhibitory effect of fat accumulation by THY, the degree of fat pinching was confirmed through Oil Red O staining in CE and tested with THY (CE data not shown). Fat accumulation was induced normally by intracellular OA (500 µM) treatment alone. As shown in Figure 3, suppression of fat accumulation was observed in the THY treatment group and, in particular, THY showed a significant fat suppression effect at 200 µM.

Effects on TG and TC Levels in HepG2 Cells
To investigate the biochemical changes induced by THY in OA-treated HepG2 cells, the triacylglycerol (TG) and total cholesterol (TC) kit was used. A normal increase in TG and TC levels

Effects on TG and TC Levels in HepG2 Cells
To investigate the biochemical changes induced by THY in OA-treated HepG2 cells, the triacylglycerol (TG) and total cholesterol (TC) kit was used. A normal increase in TG and TC levels was observed in the normal and OA-induced groups. Both the TG and TC levels were lower than that of the OA (500 µM) in the presence of THY 100 (100 µM) and THY 200 (200 µM). The THY (200 µM) level was lower than the SM (20 µg) level ( Figure 4).

Effects on TG and TC Levels in HepG2 Cells
To investigate the biochemical changes induced by THY in OA-treated HepG2 cells, the triacylglycerol (TG) and total cholesterol (TC) kit was used. A normal increase in TG and TC levels was observed in the normal and OA-induced groups. Both the TG and TC levels were lower than that of the OA (500 μM) in the presence of THY 100 (100 μM) and THY 200 (200 μM). The THY (200 μM) level was lower than the SM (20 μg) level ( Figure 4). Total intracellular triglyceride and total cholesterol were analyzed using the enzymatic colorimetric method. ## p < 0.01, relative to normal; * p < 0.05, ** p < 0.01, relative to OA. N: normal; OA: oleate; CE: C. longa 50% EtOH extract; SM: silymarin; THY: thymol.

Effects on Hepatic Lipid Accumulation and Protein Expression
Western blot analysis was performed to determine the expression of adipogenic transcription factors and enzymes in order to demonstrate the effect of THY on the protein level and lipid accumulation. Lipid accumulation induced by OA was treated with SM (20 μg), THY 100 (100 μM), and THY 200 (200 μM). Increased protein expression was observed with OA (500 μM) treatment, and Total intracellular triglyceride and total cholesterol were analyzed using the enzymatic colorimetric method. ## p < 0.01, relative to normal; * p < 0.05, ** p < 0.01, relative to OA. N: normal; OA: oleate; CE: C. longa 50% EtOH extract; SM: silymarin; THY: thymol.

Effects on Hepatic Lipid Accumulation and mRNA Expression
We determined the underlying mechanisms of SM-, THY 100-, and THY 200-induced expression of genes that are important for lipid metabolism. Genes involved in lipid production (SREBP-1c, FAS, C/EBP, and PPARγ) were significantly increased in OA-treated HepG2 cells. As shown in Figure

Effects on AMPK Expression in HepG2 Cells
AMP-activated protein kinase (AMPK) is a major regulator of fat production and fatty acid oxidation in metabolic tissues. Changes in AMPK expression in HepG2 cells are strongly linked to intracellular lipid metabolism. Therefore, HepG2 cells were treated with OA (500 μM), followed by treatment with SM (20 μg) and THY (200 μM) for 24 h for investigating the effect of THY on the phosphorylation of AMPK. As can be seen in Figure 7, THY stimulated AMPK threonine 172 phosphorylation.

Effects on AMPK Expression in HepG2 Cells
AMP-activated protein kinase (AMPK) is a major regulator of fat production and fatty acid oxidation in metabolic tissues. Changes in AMPK expression in HepG2 cells are strongly linked to intracellular lipid metabolism. Therefore, HepG2 cells were treated with OA (500 µM), followed by treatment with SM (20 µg) and THY (200 µM) for 24 h for investigating the effect of THY on the phosphorylation of AMPK. As can be seen in Figure 7, THY stimulated AMPK threonine 172 phosphorylation.

Discussion
Usually, most of the C. longa studies have focused on the components of curcumin derivatives. The aim of this study is to investigate the effects of fatty liver inhibition on NAFLD in relation to the anti-hyperlipidemic efficacy of THY, which is a minor component in C. longa. HepG2 cells derived from human liver cancer cells were used as an NAFLD model by inducing lipid accumulation with OA. When compared with the normal group, an increase in lipid accumulation was observed in the treated group. Various concentrations of THY were tested for cytotoxicity, and the results were negative. In order to directly identify the decrease in the fat content, the Oil Red O experiment was conducted, and the fat significantly decreased in the presence of THY 200. In addition, triglyceride levels and total cholesterol were inhibited. Western blot and real-time RT-PCR were conducted to verify changes in the accumulation of lipid-related factors in HepG2 cells induced by OA (Table 1). SREBP-1c is a necessary component for controlling cholesterol metabolism by protein decomposition and [14] increases gene expression associated with fat acid biosynthesis and TG maturation, such as FAS and ACC [15,16]. FAS is an enzyme that catalyzes fatty acid synthesis and converts nutrients from the liver to fat for nutrient over urban energy storage [17]. ACC is synthesized by malonyl-CoA to bind acetyl coenzymes and produce fatty acids. SREBP-1c, FAS, and ACC levels were significantly decreased by THY treatment. These results show that THY is related to the reduced extent of fat formation in OA-induced fat production. PPARγ is a transcription factor mainly expressed in adipose tissue and is an important factor in adipose tissue differentiation, and it is also involved in glucose constancy and placenta development [18]. Activation of PPARγ led to an increase in adipose tissues, and THY reduced the activation of PPARγ. C/EBP is a gene associated with cell proliferation and adipose tissue differentiation. Inhibition of C/EBP was observed in the THY treatment group. AMPK inhibits the expression of factors such as SREBP-1c, and cholesterol and fatty acid synthesis, through the oxidation of glucose and fatty acids [19,20]. The fact that THY also increased AMPK phosphoric acidification in our results can be seen as THY inhibiting lipid accumulation through increased AMPK phosphorylation. In summary, the underlying mechanism of THY-induced inhibition of nonalcoholic fatty liver disease involves AMPK activation, and activated AMPK further inhibits the synthesis of SREBP-1c and ACC. Inhibited SREBP-1c restrains FAS, and inhibited proteins further inhibit non-alcoholic fatty liver disease by reducing fatty acid, cholesterol, and triglyceride synthesis. In addition, it can be used for the quantitative and qualitative determination of THY, which is useful in improving the quality control of ethanolic extract from C. longa and its pharmaceutical preparations.

Discussion
Usually, most of the C. longa studies have focused on the components of curcumin derivatives. The aim of this study is to investigate the effects of fatty liver inhibition on NAFLD in relation to the anti-hyperlipidemic efficacy of THY, which is a minor component in C. longa. HepG2 cells derived from human liver cancer cells were used as an NAFLD model by inducing lipid accumulation with OA. When compared with the normal group, an increase in lipid accumulation was observed in the treated group. Various concentrations of THY were tested for cytotoxicity, and the results were negative. In order to directly identify the decrease in the fat content, the Oil Red O experiment was conducted, and the fat significantly decreased in the presence of THY 200. In addition, triglyceride levels and total cholesterol were inhibited. Western blot and real-time RT-PCR were conducted to verify changes in the accumulation of lipid-related factors in HepG2 cells induced by OA (Table 1). SREBP-1c is a necessary component for controlling cholesterol metabolism by protein decomposition and [14] increases gene expression associated with fat acid biosynthesis and TG maturation, such as FAS and ACC [15,16]. FAS is an enzyme that catalyzes fatty acid synthesis and converts nutrients from the liver to fat for nutrient over urban energy storage [17]. ACC is synthesized by malonyl-CoA to bind acetyl coenzymes and produce fatty acids. SREBP-1c, FAS, and ACC levels were significantly decreased by THY treatment. These results show that THY is related to the reduced extent of fat formation in OA-induced fat production. PPARγ is a transcription factor mainly expressed in adipose tissue and is an important factor in adipose tissue differentiation, and it is also involved in glucose constancy and placenta development [18]. Activation of PPARγ led to an increase in adipose tissues, and THY reduced the activation of PPARγ. C/EBP is a gene associated with cell proliferation and adipose tissue differentiation. Inhibition of C/EBP was observed in the THY treatment group. AMPK inhibits the expression of factors such as SREBP-1c, and cholesterol and fatty acid synthesis, through the oxidation of glucose and fatty acids [19,20]. The fact that THY also increased AMPK phosphoric acidification in our results can be seen as THY inhibiting lipid accumulation through increased AMPK phosphorylation. In summary, the underlying mechanism of THY-induced inhibition of non-alcoholic fatty liver disease involves AMPK activation, and activated AMPK further inhibits the synthesis of SREBP-1c and ACC. Inhibited SREBP-1c restrains FAS, and inhibited proteins further inhibit non-alcoholic fatty liver disease by reducing fatty acid, cholesterol, and triglyceride synthesis. In addition, it can be used for the quantitative and qualitative determination of THY, which is useful in improving the quality control of ethanolic extract from C. longa and its pharmaceutical preparations.  SREBP-1c  CGC AAG GCC ATC GAC TAC AT  GAC TTA GGT TCT CCT GCT TGA GTT TC  FAS  AAG GAC CTG TCT AGG TTT GAT GC  TGG CTT CAT AGG TGA CTT CCA  ACC  TCG CTT TGG GGG AAA TAA AGT G  ACC ACC TAC GGA TAG ACC GC  C/EBP  CAA GCA CAG CGA CGA GTA CAA  GCT TGA ACA AGT TCC GCA GGG  PPARγ  GCA GGC TCC ACT TTG ATT  ACC ACT CCC ACT CCT TTG  GAPDH  CAG GGC TGC TTT TAA CTC TGG T  GAT TTT GGA GGG ATC TCG CT

Extraction and Isolation Procedure
The dried and sliced C. longa (1 kg) were extracted twice with 80% aqueous MeOH (5 L) at room temperature. The extracts were successively partitioned with water (1 L), ethyl acetate (EtOAc) (1 L × 3), and n-butanol

High-Performance Liquid Chromatography Analysis
The dried and sliced C. longa roots were homogenized using a ball mill (Retsch MM400, Haan, Germany). Fine powder, which was passed through a 40-mesh sieve, was weighed (1000 mg), extracted in 5 mL of 50% fermented EtOH, and ultrasonically extracted for 1 h at 35 • C. The extract was then evaporated under reduced pressure. The extract solution was filtered through a syringe filter (0.22 µm) and injected directly into the HPLC system. HPLC was carried out on a 1200-RRLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary solvent delivery system and an autosampler. Chromatographic separations were carried out a YMC Pack ODS-AM column (4.6 × 250 mm, 5 µm). The column oven was maintained at 35 • C, and the mobile phases were composed of solvent A (water containing 0.1% (vol/vol) formic acid) and solvent B (acetonitrile containing 0.1% (vol/vol) formic acid). Elution conditions of liquid chromatography, which were optimized, were as follows: 0-3 min, 70% B; 3-16 min, 50% B; and 16-20 min, 30% B. The flow rate was 1 mL/min. A 10 µl aliquot was injected into the column using the autosampler.

Cell Culture
The human hepatocellular carcinoma cell line for HepG2 cells was purchased from the American Tissue Culture Collection (ATCC, catalog no. HB-8065; Manassas, VA, USA) and were cultured in DMEM and 10% FBS. The HepG2 cells were incubated at 37 • C under 95% humidified atmosphere and 5% CO 2 .

Cell Viability
Cell viability was achieved through the MTS assay. HepG2 cells were seeded at a density of 5 × 10 5 cells/mL in 24-well cell culture plates (Nunc, A/S, Roskilde, Denmark). A day after incubation, a plate was tested in two parts. Cells were treated with various concentrations of CE (200 µg), SM (20 µg), THY (50, 100, 200, and 400 µM), and OA (500 µM). The plates were incubated for 18 h at 37 • C under 5% CO 2 . The existing medium was replaced with a new medium supplemented with an MTS solution (5 mg/mL). After 20 min, a microplate reader (BioTek South Korea, Seoul, Korea) was used to automatically read the absorbance of each well at 490 nm.

Measurement of Lipid Levels
HepG2 cells were cultured in a 24-well plate at a density of 4 × 10 5 cells/mL for a day. Then, the plate was treated with CE (200 µg), SM (20 µg), and THY (100, 200 µM) with OA (500 µM). TC and TG levels were measured according to the kit protocol (BioVision, Mountain View, CA, USA). At the end of the experiment, the absorbance of each well was recorded at 450 nm using a microplate reader.

Western Blot Analysis
The HepG2 cells were harvested in a six-well plate at a density of 5 × 10 5 cells/mL. Subsequently, the cells were treated with SM (20 µg) and THY (100, 200 µM). One hour later, OA (500 µM) was added for 24 h in a 37 • C incubator with a 95% humidified atmosphere and 5% CO 2 . After 24 h of incubation, the cells were washed twice with DPBS. Next, the HepG2 cells were lysed with a lysis buffer for 20 min. For protein extraction, centrifugation was performed at 16,000× g at 4 • C for 5 min. Protein samples were quantified according to the quantification protocol, and the quantified samples were SDS gel electrophoresed. The running gel was transferred to polyvinylidene difluoride (PVDF; Millipore, Bedford, MA, USA) membranes at 15 V for 45 min. The membranes were blocked with 3% BSA for 1 h and left overnight at 4 • C with primary antibodies against SREBP-1c (1:1000), FAS (1:1000), ACC (1:1000), C/EBP (1:1000), PPARγ (1:1000), pAMPK(1:1000), and AMPK(1:1000). Subsequently, the membrane was washed several times in TBST and treated with secondary antibodies (1:5000) at Processes 2020, 8, 1191 9 of 10 room temperature. Washed membranes were detected using Atto ECL plus (Tokyo, Japan). An Image Quant LAS 4000 Mini Bio molecular Imager (GE Healthcare, UK) was used for analyzing the bands.

Real-Time RT-PCR
Total RNA was isolated using an E.Z.N.A. ® Total RNA Kit (Omega Bio-tek, Inc.; Norcross, GA, USA) following the kit protocol. Reverse transcription was performed using a QuantiTect ® Reverse Transcription Kit (Qiagen, Hilden, Germany) and quantitative real-time RT-PCR was performed using a Power SYBR ® Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc. Foster City, CA, USA) according to protocol.

Statistical Analysis
All data were collected through statistical analysis, followed by Duncan's test for various comparisons. The results are presented as a mean ± standard deviation of three independent experiments. All calculations were performed using SPSS Statistics 23 software (SPSS Inc., Chicago, IL, USA). Comparison with * p < 0.05 or ** p < 0.01 was considered statistically significant.