Phenolic Compounds with Antioxidant Properties from Canola Meal Extracts Inhibit Adipogenesis

The extraction of phenolic compounds from canola meal produces functional health products and renders the canola meal a more digestible animal feed. The extracted phenolics may have novel bioactivity worth investigation. In this study, several solvents were evaluated for their ability to extract phenolic compounds from canola meal: water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The in vitro antioxidant and anti-obesity properties of various extracts were investigated. Anti-obesity properties were studied using adipogenic differentiation inhibition of a murine mesenchymal stem cell line (C3H10T1/2) and a pancreatic lipase inhibition assay. AE, ME, and BE showed significant (p < 0.05) adipogenesis and pancreatic lipase inhibitory activities and may have more pharmacological properties. AE down-regulated the gene expression of the major adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), correlating to phenolic content in a dose-dependent manner. The chemical characterization of AE revealed the presence of sinapic acid, ferulic acid, and kaempferol derivatives as main bioactive phenols.


Introduction
Canola (Brassica napus L., rapeseed, oilseed rape) is used as a medicinal food in Middle Asia, North Africa, and West Europe. There is also evidence of its use by early Australian and New Zealand settlers as well as the indigenous population [1]. Large amounts of protein-rich canola meal are generated globally during the extraction of canola seed oil. Over 70 million tonnes of canola/oilseed rape is produced per year, resulting in over 40 million tonnes of canola meal. The high phenolic content of canola meal renders it less appealing as an animal feed. On the other hand, plant phenols are attracting increasing attention as multipotent antioxidant molecules that can be used to generate high-value nutraceutical products. The extraction of canola meal phenols is an appealing high-tech solution to generate antioxidant-rich extracts for the food and pharmaceutical industries while enhancing the value of the remaining meal by increasing its digestibility [2].
Polyphenols are a major component of the bioactive molecules isolated from plant extracts and have been associated with their ability to modify a range of diseases, including cardiovascular Sinapic acid [27], and hydroxycinnamic acids [28] are known for the lipolysis and their role in anti-adipogenesis. Polyphenols including quercetin, resveratrol, apigenin, and myricetin are known to bind with particular residues (Phe264, His266, Ile281, Cys285, and Met348) of the PPARγ receptor to establish anti-adipogenicity during the early stages of the differentiation [29]. For example, ethanol extracts of bamboo stems have been shown to play a vital role in the downregulation of differentiation markers C/EBPβ, PPARγ, and FABP4 (fatty acid-binding protein) [30]. Likewise, extracts of Mangifera indica L. leaf have also been shown to be effective in reducing the effect of PPARγ suggesting their potential use in obesity management [31]. Furthermore, polyphenol extracts of purple maize (Zea mays L) and purple silk corn extracts have been shown to have activities such as anti-inflammatory,anti-adipogenic,anti-diabetic and induction of lipolysis [32,33]. Moreover, pomegranate juice has also been shown to down-regulate adipogenic genes and lipase [34]. Specific compounds identified include quercetin, luteolin, vanillic acid and protocatechuic acid, rutin [33], ellagic acid and punicalagin [34].
Pancreatic lipase is a key enzyme secreted from the pancreas and plays a major role in the hydrolysis of 50%-70% of dietary triglycerides to monoacylglycerides and free fatty acids before absorption by enterocytes [20]. Therefore, inhibition of this enzyme may result in lower fat absorption. In this study, various solvent mixtures were used to recover canola meal phenols and study their composition and bioactivities. The phenolic composition of various extracts from canola meal, their in vitro antioxidant and anti-obesity activities were investigated.

Results
Canola meal was extracted with water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The canola meal extracts CME were chemically characterized and screened for their anti-obesity properties.

Phenolic Composition of Canola Meal Extracts
Gallic acid was used to produce a calibration curve by linear regression for the analysis of total phenolic content (TPC) from canola meal extracts (R 2 = 0.9998). The TPC of various extracts in decreasing order was as follows: AE > BE > ME > EE > WE > CE > HE (Figure 1). to bind with particular residues (Phe264, His266, Ile281, Cys285, and Met348) of the PPARγ receptor to establish anti-adipogenicity during the early stages of the differentiation [29]. For example, ethanol extracts of bamboo stems have been shown to play a vital role in the downregulation of differentiation markers C/EBPβ, PPARγ, and FABP4 (fatty acid-binding protein) [30]. Likewise, extracts of Mangifera indica L. leaf have also been shown to be effective in reducing the effect of PPARγ suggesting their potential use in obesity management [31]. Furthermore, polyphenol extracts of purple maize (Zea mays L) and purple silk corn extracts have been shown to have activities such as anti-inflammatory,anti-adipogenic,anti-diabetic and induction of lipolysis [32,33]. Moreover, pomegranate juice has also been shown to down-regulate adipogenic genes and lipase [34]. Specific compounds identified include quercetin, luteolin, vanillic acid and protocatechuic acid, rutin [33], ellagic acid and punicalagin [34]. Pancreatic lipase is a key enzyme secreted from the pancreas and plays a major role in the hydrolysis of 50%-70% of dietary triglycerides to monoacylglycerides and free fatty acids before absorption by enterocytes [20]. Therefore, inhibition of this enzyme may result in lower fat absorption. In this study, various solvent mixtures were used to recover canola meal phenols and study their composition and bioactivities. The phenolic composition of various extracts from canola meal, their in vitro antioxidant and anti-obesity activities were investigated.

Results
Canola meal was extracted with water (WE) and various 80% organic solvent/water mixtures of methanol (ME), acetone (AE), ethanol (EE), butanol (BE), chloroform (CE) and hexane (HE). The canola meal extracts CME were chemically characterized and screened for their anti-obesity properties.

Phenolic Composition of Canola Meal Extracts
Gallic acid was used to produce a calibration curve by linear regression for the analysis of total phenolic content (TPC) from canola meal extracts (R 2 = 0.9998). The TPC of various extracts in decreasing order was as follows: AE > BE > ME > EE > WE > CE > HE ( Figure 1).  Various extracts were chemically characterized by HPLC-DAD-MS/MS using commercially available reference standards and spectral data from previous literature [2]. Different extracts showed different phenolic profile [35] that reflects the relative polarity of various extracting solvents. In accord with total phenolic content, the acetone extract AE showed the largest number of peaks and the highest recovery (peak height). Most peaks were of intermediate polarity, eluting between 15-40 min in the 60-minute long chromatograms ( Figure 2). In alcoholic and acetone extracts, the major peak observed was sinapine (Peak 1). The major peak observed in water, chloroform, and hexane extracts was feruloyl choline (4-O-8') guaiacyl-di-sinapoyl (Peak 7). Various extracts were chemically characterized by HPLC-DAD-MS/MS using commercially available reference standards and spectral data from previous literature [2]. Different extracts showed different phenolic profile [35] that reflects the relative polarity of various extracting solvents. In accord with total phenolic content, the acetone extract AE showed the largest number of peaks and the highest recovery (peak height). Most peaks were of intermediate polarity, eluting between 15-40 min in the 60-minute long chromatograms ( Figure 2). In alcoholic and acetone extracts, the major peak observed was sinapine (Peak 1). The major peak observed in water, chloroform, and hexane extracts was feruloyl choline (4-O-8') guaiacyl-di-sinapoyl (Peak 7). Compounds present in AE were characterized by HPLC-DAD with online detection of free radical scavenging activity of eluting compounds using an ABTS scavenging assay, as shown in Figure 2, Table 1. Peaks 1-9 in Figure 2 are the main contributors to the antioxidant activity of the extract (showing peaks in the ABTS chromatogram).  Compounds present in AE were characterized by HPLC-DAD with online detection of free radical scavenging activity of eluting compounds using an ABTS scavenging assay, as shown in Figure 2, Table 1. Peaks 1-9 in Figure 2 are the main contributors to the antioxidant activity of the extract (showing peaks in the ABTS chromatogram).  Compounds present in AE were characterized by HPLC-DAD with online detection of free radical scavenging activity of eluting compounds using ABTS scavenging assay as shown in Fig. 2, Table;1. Peaks 1-9 in Fig. 2 are the main contributors to the antioxidant activity of the extract (showing peaks in the ABTS chromatogram).  Compounds present in AE were characterized by HPLC-DAD with online detection of free radica activity of eluting compounds using ABTS scavenging assay as shown in Fig. 2, Table;1. Peaks 1-9 in Fig. 2 contributors to the antioxidant activity of the extract (showing peaks in the ABTS chromatogram). , no peak; RT, retention time; λ max , UV-vis spectra; ESI − , electrospray ionization peaks in negative mode; ESI + , electrospray ionization peaks in positive mode; MW, molecular weight; NI, did not ionize under ESI modes; b, broad peak; s, peak shoulder; ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) representing ABTS scavenging activity in online assay (antioxidant); +, low ABTS scavenging activity; ++, good ABTS scavenging activity; +++, high ABTS scavenging activity.

Canola Meal Extracts Inhibit Intracellular Lipid Accumulation
Cell viability was determined by the CellTiter 96 ® AQueous non-radioactive cell proliferation assay according to the manufacturer's protocol [14]. The CellTiter 96 ® AQueous assay constitutes of a solution of a novel [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and an electron coupling reagent (phenazine methosulfate, PMS). MTS is bio-reduced by cells into a formazan product that is soluble in tissue culture medium. Untreated cells served as a control and were assumed to be 100% viable. To assess the effect of DMSO on the viability, cells were incubated with various concentrations of DMSO (0.05%-0.3%) in cell culture medium, DMEM, and the proportion of viable cells was determined ( Figure 3A). The toxicity of various extracts on cells was examined in the range 1-3 mg/mL ( Figure 3B).

Canola Meal Extracts Inhibit Intracellular Lipid Accumulation
Cell viability was determined by the CellTiter 96 ® AQueous non-radioactive cell proliferation assay according to the manufacturer's protocol [14]. The CellTiter 96 ® AQueous assay constitutes of a solution of a novel [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and an electron coupling reagent (phenazine methosulfate, PMS). MTS is bio-reduced by cells into a formazan product that is soluble in tissue culture medium. Untreated cells served as a control and were assumed to be 100% viable. To assess the effect of DMSO on the viability, cells were incubated with various concentrations of DMSO (0.05%-0.3%) in cell culture medium, DMEM, and the proportion of viable cells was determined ( Figure 3A). The toxicity of various extracts on cells was examined in the range 1-3 mg/mL ( Figure 3B).  The in vitro anti-adipogenic effect of CME was examined with 1.5 mg/mL of EE and BE, and 2 mg/mL of AE, ME, HE, CH, and WE in the presence of adipogenic differentiation media (ADM) for seven days. Cells of C3H10T1/2 were then stained with Oil Red O stain ( Figure 4A) and visualized by light microscopy and quantified by measuring absorbance at 510 nm ( Figure 4B). Fat globules appeared as red granules, as shown in Figure 4A, demonstrating differentiation into adipocytes [36]. Following staining with Oil Red O, a clear distinction can be seen in undifferentiated cells ( Figure 4A, panel i) and differentiated cells ( Figure 4A, panel ii). are expressed as mean ± standard deviation (n = 3). Bars with different letters have mean values that are significantly different (p < 0.05) at the same concentration.
The in vitro anti-adipogenic effect of CME was examined with 1.5 mg/mL of EE and BE, and 2 mg/mL of AE, ME, HE, CH, and WE in the presence of adipogenic differentiation media (ADM) for seven days. Cells of C3H10T1/2 were then stained with Oil Red O stain ( Figure 4A) and visualized by light microscopy and quantified by measuring absorbance at 510 nm ( Figure 4B). Fat globules appeared as red granules, as shown in Figure 4A, demonstrating differentiation into adipocytes [36]. Following staining with Oil Red O, a clear distinction can be seen in undifferentiated cells ( Figure  4A, panel i) and differentiated cells ( Figure 4A, panel ii).  All extracts inhibited stem cell differentiation to some extent. The cells treated with AE showed the highest level of inhibition (p < 0.05). In general, we can categorize the CME into the following clusters: strong inhibitors (AE and HE), moderate inhibitors (ME, BE, and WE), and mild inhibitors (EE and CE). Accordingly, large lipid vacuoles were clearly noticeable in EE-and CE-treated cells, showing minimal inhibition had occurred ( Figure 4A, panels vi,viii).
Excess storage of lipid as adipose tissue has been identified as a risk factor for the development of many diseases [37]. Although canola meal has never been studied for its anti-adipogenic potential, many naturally-occurring flavonoids, phenolic acids, and lignans have demonstrated a capacity to inhibit the lipid droplet deposition in the adipose tissue [38].

Canola Meal Extract Mediated Reductions in Lipid Accumulation are Correlated with a Reduction in PPARγ Expression
To determine the effects of CMEs on PPARγ expression, PPARγ immunostaining of cells undergoing differentiation was undertaken ( Figure 5A,B). The typical nuclear localization of PPARγ was seen in differentiated cells ( Figure 5B, panel DC). No nuclear PPARγ staining was observed for either AEor BE-treated cells, and a low level of staining was observed in HE-treated cells. BE appeared to inhibit adipogenic differentiation at the lower dose of 1.5 mg/mL. These results indicate that CMEs appear to differ in their ability to inhibit adipogenesis to different extents. The level of PPARγ gene expression in the cells treated with CMEs was determined using qRT-PCR ( Figure 5B). Cells incubated in culture media alone acted as a negative control and untreated fully-differentiated cells were used as a positive control. The expression level of PPARγ in the C3H10T1/2 cells treated with AE exhibited a significant decrease (p < 0.05) compared to the positive control. BE treated cells also showed a lower expression of PPARγ, and a reducing trend was observed as follows: HE < ME < WE < EE < CE. PPARγ gene expression levels followed the same trend seen with Oil Red O staining ( Figure 4A) and PPARγ immunofluorescence staining ( Figure 5A).
Previous studies have shown that reduced PPARγ gene expression correlates with inhibition of adipogenesis in 3T3-L1 adipocyte cells treated with extracts of cranberries and onion peel [22,39]. Also, canola proteins and their hydrolysates have been shown to reduce the expression of genes coding for PPARγ proteins in C3H10T1/2 cells [14].

Inhibition of Pancreatic Lipase Activity
The IC 50 values for pancreatic lipase (PL) inhibition of the seven CME are shown in Figure 6. All extracts demonstrated some level of inhibition towards PL with AE showing the highest (1.60 ± 0.06 mg/mL), and the CE the lowest (5.42 ± 0.07 mg/mL) (p < 0.05) levels of inhibition. BE also showed a high level of PL inhibition. From these results, we conclude that the phenolic compounds in AE and BE are active in both the inhibition of lipid accumulation in adipocytes and lipase enzymatic activity. Several plant extracts have been shown to have PL inhibitory effects [40]. Previous studies have shown that reduced PPARγ gene expression correlates with inhibition of adipogenesis in 3T3-L1 adipocyte cells treated with extracts of cranberries and onion peel [22,39]. Also, canola proteins and their hydrolysates have been shown to reduce the expression of genes coding for PPARγ proteins in C3H10T1/2 cells [14].

Inhibition of Pancreatic Lipase Activity
The IC50 values for pancreatic lipase (PL) inhibition of the seven CME are shown in Figure 6. All extracts demonstrated some level of inhibition towards PL with AE showing the highest (1.60 ± 0.06 mg/mL), and the CE the lowest (5.42 ± 0.07 mg/mL) (p < 0.05) levels of inhibition. BE also showed a high level of PL inhibition. From these results, we conclude that the phenolic compounds in AE and BE are active in both the inhibition of lipid accumulation in adipocytes and lipase enzymatic activity. Several plant extracts have been shown to have PL inhibitory effects [40].

Discussion
Acetone showed superior phenol extraction abilities (287.7 ± 16.3 mg GAE/g extractable matter), which has been observed before in other plant extracts [41]. Butanol and methanol extracts showed high levels of extraction, without a significant difference in their abilities to extract canola phenolic compounds. Although ethanol has an intermediate polarity between methanol and butanol, ethanol extracts recovered significantly less phenols. Highly-polar (water) and non-polar (chloroform and hexane) solvents showed the least potential to extract canola phenols. Phenolic profiling of all extracts

Discussion
Acetone showed superior phenol extraction abilities (287.7 ± 16.3 mg GAE/g extractable matter), which has been observed before in other plant extracts [41]. Butanol and methanol extracts showed high levels of extraction, without a significant difference in their abilities to extract canola phenolic compounds. Although ethanol has an intermediate polarity between methanol and butanol, ethanol extracts recovered significantly less phenols. Highly-polar (water) and non-polar (chloroform and hexane) solvents showed the least potential to extract canola phenols. Phenolic profiling of all extracts was undertaken [35]; however, only the chemical characterization of the extract with the highest phenol content (AE) is discussed here. Alcohol and acetone extracts showed the largest number of peaks.
Interestingly, water extracts were comparable to hexane and chloroform extracts in their ability to extract phenolic compounds as these extracts contained almost the same number of peaks. However, water extracts appeared to have higher recovery. The data gathered from UV-vis spectra, ABTS scavenging activity, and relative retention times were compared with reference standards and literature data to characterize the chemical composition of CMEs as described previously [2]. The online-ABTS HPLC analysis showed that, generally, most detected compounds demonstrate good ABTS radical scavenging activity. A few exceptions can be observed, such as Peak 5 (kaempferol-sinapoyl-trihexoside), which had no ABTS scavenging activity. Meanwhile, Peak 2 (feruloyl choline guiacyl) was the sixth most abundant component in AE (Figure 2A), yet it appeared as the second highest peak in the online ABTS radical scavenging trace ( Figure 2B), reflecting a strong free radical scavenging activity that is not proportional to its relative concentration.
DMSO (0.2%) solution was selected as the solvent that achieved a complete dissolution of extracts and maintained approximately 70% cell viability in accord with previous studies [42]. The anti-adipogenic effects of samples at these concentrations has been shown not to be due to the effect of DMSO [43]. All extracts demonstrated more than 70% viability at ≤ 2 mg/mL, apart from BE and EE which showed similar viability at ≤ 1.5 mg/mL. Therefore, 1.5 mg/mL of the ethanol and butanol extracts, and 2 mg/mL of all other extracts were used for cell culture experiments ( Figure 3B).
All extracts demonstrated significant inhibition of stem cell differentiation. The cells treated with AE showed the highest inhibition (p < 0.05). In general, we can categorize the CME into the following clusters: strong inhibitors (AE and HE), moderate inhibitors (ME, BE, and WE), and mild inhibitors (EE and CE). Accordingly, large lipid vacuoles were clearly noticeable in EE-and CE-treated cells showing minimal inhibition had occurred ( Figure 4A, panels vi,viii).
The inhibition of PL has been identified as a potential target for the treatment of obesity. So far, many plants have been examined for their PL inhibitory potential [40]. All CMEs showed some PL inhibitory potential ( Figure 6). The most potent inhibitor was AE followed by alcoholic extracts, while the least potent were HE and CE. Lipase inhibition correlated well with the degree of adipogenic inhibition measured by Oil Red O staining ( Figure 4A).
The murine embryonic fibroblast mesenchymal stem cell line (C3H10T1/2) was purchased from the American Type Culture Collection (Rockville, MD, USA). The CellTitre®AQueous non-radioactive cell proliferation assay kit and GoTaq Green 2× master mix were purchased from Promega (Fitchburg, WI, USA); Aurum™ total RNA kit and iScript™ advance cDNA synthesis kits were obtained from Bio-Rad Laboratories (Hercules, CA, USA); and RT-PCR grade water was purchased from Life Technologies (Scoresby Victoria, Australia).

Recovery of Canola Meal Biophenols
Canola meal was extracted with water and a range of aqueous 80% solvents, namely, acetone, methanol, butanol, ethanol, hexane, and chloroform. Canola meal phenols are essentially hydrophilic in nature [44][45][46]. Using hydro-organic solvents maximizes the potential of extracting canola phenols both qualitatively and quantitively. We employed various solvents with a wide range of polarities in order to comprehensively explore canola meal bioactive constituents. While hydro-alcoholic solvents are the most commonly used extraction solvent for plant phenols [47,48], hydro-acetone solvents showed superior quantitative properties [2,35]. Chloroform and hexane are immiscible with water, while n-butanol has limited water miscibility. Their mixtures with water form interesting two-phase extraction systems that have been frequently reported in the literature [49,50]. Chemical characterization of the extracts has been performed [35]. The prepared extracts were freeze-dried and referred to as acetone extract (AE), methanol (ME), butanol (BE), ethanol (EE), hexane (HE), chloroform (CE), and water (WE). All freeze-dried extracts were then reconstituted in 50% methanol and filtered through a syringe filter (0.22 µm).

Measurement of Total Phenolic Content
Total phenolic content (TPC) was determined using the Folin-Ciocalteu reagent, as previously described [2]. A calibration curve was produced using a range of gallic acid concentrations (0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/mL) in 50% aqueous methanol. A 100 µL aliquot of each standard or sample was added to a 10 mL volumetric flask, containing 7 mL of ultra-pure water (UPW) and then mixed with 500 µL of Folin-Ciocalteu reagent and 1.5 mL of 20% sodium carbonate. Following incubation for 1 h at room temperature, the absorbance was measured at 760 nm using a Cary 50 spectrophotometer (Varian, Victoria, Australia), with software Cary WinUV version 3, (Varian, Victoria, Australia), and results were expressed as milligrams of gallic acid equivalents (GAE) per gram dry weight (mg GAE/g DW).

Cell Culture and Adipogenic Differentiation
C3H10T1/2 cells were cultured in DMEM, containing 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine and incubated at 37 • C in a humidified incubator with 5% CO 2 . Media was replaced every 3 days. At 75% confluency, cells were trypsinized using 0.25% trypsin-EDTA solution for 2-5 min and then re-suspended in DMEM cell culture medium in a 1:10 ratio. Cell viability was assessed using the CellTiter 96®AQueous non-radioactive cell proliferation assay, according to the manufacturer's protocol, with C3H10T1/2 cells plated at a density of 5000 cells/well in a 96-microplate cultured first with 0.1% to 0.3% DMSO with DMEM. Then, depending on the solubility of each extract, samples were used at a percentage of 1% to 3% with a suitable DMSO percentage. DMEM was used as a negative control. Following incubation for 24 h at 37 • C, absorbance was measured at 570 nm using a FLUOstar omega UV-vis spectrophotometer (BMG Labtech, Offenburg, Germany).
ADM was replaced every 48 h, for a seven-day period before the cells were examined by Oil Red O staining.

Oil-Red O Staining Staining and Quantification of Intracellular Lipid Droplets
Oil Red O staining was performed according to the procedure described previously [51]. Briefly, C3H10T1/2 cells were treated with ADM containing 2 mg/mL of AE, ME, HE, CE, and WE or 1.5 mg/mL of BE and EE extracts for seven days. Cells were rinsed briefly with PBS, fixed, and air-dried before staining, then rinsed carefully with PBS, and viewed under an inverted microscope (Nikon Eclipse Ti-U inverted, Japan).

Immunofluorescence Staining of PPARγ
Cells were incubated with canola extracts (as detailed above) in ADM for seven days, after this period cells were fixed with 3% paraformaldehyde (PFA), rinsed with phosphate buffer saline (PBS), then treated with 0.1% Triton X-100 for 7 min at room temperature (RT), and rinsed again with PBS. The cells were then incubated for 30 min in blocking buffer prepared by mixing 5% goat serum (Gibco ® , Eggenstein, Germany) in PBS. The cells were then incubated with anti-PPARγ (81B8) rabbit monoclonal antibody (1:50) Cell Signalling Technology (Danvers, MA, USA) for one hour at ambient temperature, then washed gently with PBS and incubated in the dark with anti-rabbit IgG (Fab 2)-Alexa Fluor ® 488 (1:100; Cell Signalling Technology) for one hour. Finally, they were counterstained with 4',6-diamidino-2-phenylindole (DAPI) counterstain. Cells were observed using an A1R + /A1 + confocal laser microscope system (Nikon, NY, USA).

Quantitative PCR (qPCR) of PPARγ Gene Expression
Total RNA was isolated from treated and un-treated C3H10T1/2 cells using the Aurum™ total RNA kit (Bio-Rad), following the manufacturer's protocol. The concentration and quality of RNA were measured using a Nanodrop 2000'analyser (Thermo Scientific Ltd, Melbourne, Australia). The purity of the RNA samples was measured and an absorbance ratio at A260/A280 of 1.84 was achieved.
Complementary DNA (cDNA) was synthesized using the iScript Advance cDNA synthesis kit for RT-qPCR (Bio-Rad) according to the manufacturer's protocol. The reverse transcription reaction was incubated in a thermocycler using the amplification cycles at 25 • C, 5 min; 42 • C, 30 min; 85 • C, 5 min, and 4 • C, 5 min. The amplification of the synthesized cDNA was performed by PCR at a final concentration of 300 ng/20 µL, using 12.5µL 2× GoTaq buffer, with 0.5 µL (1 µM) of forward and reverse primers specific for PPARγ and β-actin, the housekeeping gene.
For real-time PCR amplification, a total volume of 20 µL reaction was prepared using 2× Soso fast mix (BioRad): 10 µL 2× Soso fast mix, 0.1 µL (at a final concentration of 0.2 µM) of forward and reverse primers, 1 µL of cDNA template (600 ng), with a total reaction volume of 20 µL with DNase-free water. The qPCR reactions were carried out using a 'C1000 thermal cycler with real-time system CFX96' (Bio-Rad) using the following parameters: initially at 95 • C, 3 min; followed by 95 • C, 1 min; 5 9 • C, 1 min; and repeated 39 times.

Pancreatic Lipase Inhibition
Inhibition of pancreatic lipase (PL) was determined as described [53]. CMEs were dissolved in Tris-HCl buffer (13 mM) containing 50 mM NaCl and 1.3 mM CaCl 2 . The reaction mixture was prepared in a 96-well microplate and included with 25 µL of each sample 50 µL of substrate [Orlistat] (0.1 mM), and 25 µL of PL enzyme (50 U/mL) and incubated at 25 • C for 30 min and the reaction was stopped with the addition of 100 µL of sodium citrate (100 mM, pH 4.2).
The relative fluorescence intensity was measured using a Cary eclipse fluorescence spectrophotometer (Varian, Inc, Victoria, Australia) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The results were expressed as an IC 50 value, obtained via a least square regression line of the logarithm of the amount of samples against the pancreatic lipase activity (%).
All CME at 1.5 mg/mL concentration in 50 % aqueous methanol were filtered through a 0.22 µm syringe filter, following vortex/sonication before being analyzed by HPLC. All conditions were maintained as described by Obied et al. [2] with minor modifications. Each sample, blanks (50% aqueous methanol), and standards were analyzed for qualitative control and identification purposes.
Online ABTS with HPLC-DAD was achieved on a Varian Prostar 240 solvent delivery system connected with a Varian Prostar 410 autosampler. In addition, the outflow from HPLC-DAD was attached to a reaction coil (PEEK; 3.4 m × 0.178 mm, maintained at 37 • C) joined to a Perkin-Elmer series 10 HPLC pump (Varian 2401 pump). Changes in ABTS •+ absorbance were measured at 414 nm using a Varian 9050 UV-vis detector. Data analysis was performed using the Star chromatography workstation version 6. For the HPLC-DAD-MS/MS, all required conditions were maintained as described by Obied et al. [2] with minor modifications. The total run time was 70 min including the MS procedure, and was performed in both the negative and positive ion mode (m/z 100−1200). Results were analyzed using an Agilent Mass Hunter workstation version B.01.04 2008 (Agilent Technologies, Waldbronn, Germany).
For quantitative determination, each extract was analyzed in triplicate at 280 nm and the mean reported. Sinapic acid (0.0625 to 1100 µg/mL) was used as the standard to generate a calibration curve for quantification (R 2 = 0.9935) and concentrations expressed as milligram of sinapic acid equivalent per gram of dry weight (mgSAE/g DW).

Statistical Analysis
Experiments were performed in triplicate, and results are presented as the mean ± standard deviation (SD). All results were analyzed using Graph pad prism 5, Microsoft Excel 2016, and one-way analysis of variance (ANOVA) using statistical analysis system (SAS ® system) for Windows V8 (SAS institute, NC, USA). Comparison between sample means were calculated using the Duncan multiple range test at a 5% probability level (p < 0.05).

Conclusions
The most abundant phenols in CMEs are in AE, ME, and BE. These extracts demonstrated potent anti-adipogenic and anti-lipase activities. At the molecular level, a marked reduction in PPARγ mRNA expression was associated with AE and BE treatment of adipogenic differentiating cells. CME showed ABTS radical scavenging activity. AE has the highest content of TP and the most potent ABTS scavenging, anti-adipogenic, and PL inhibition. Sinapine and derivatives of sinapic acid, namely, kaempferol and ferroyl choline guiacyl, are the main contributors to CME antiradical activities. The results obtained in this study demonstrate the importance of solvent choice for the recovery of biophenols and in the observed pharmacological properties. Phenols are the main active constituents responsible for antioxidant and anti-adipogenic activity. Further research is required to isolate the main bioactive phenols, study the mechanism of action and find out if these activities are reproducible in vivo.