Unripe Rubus coreanus extract with ellagic acid may increase fat breakdown and heat production in cells and living animals

As shown in chemical structure (Figure 1), we previously published the results of 5-uRCK compounds (erycibelline, 5-hydroxy-2-pyridinemethanol, p-aminophenol, m-hydroxyphenylglycine, 4-hydroxycoumarin and ellagic acid) analysis [22,24]. We reported the effects of 5-uRCK and ellagic acid on the inhibition of lipid accumulation during the differentiation of 3T3-L1 preadipocytes to adipocytes. Furthermore, our previous study reported that 5-uRCK and ellagic acid treatment did not affect the viability of 3T3-L1 preadipocytes and mature adipocytes [21,22,23]. In addition, all our previous studies have indicated that 5-uRCK has the greatest antiobesity and antihypercholesterolemic effects of all tested uRCK extracts prepared by various extraction methods (water, 5% ethanol, 30% ethanol and 70% ethanol) [21,22,23,24,25]. Quantitative analysis by HPLC showed that the aqueous extract and 5-uRCK contained 14–16 mg/g and 18–21 mg/g ellagic acid, respectively [24]. Other compounds, such as erycibelline, 5-hydroxy-2-pyridinemethanol, m-hydroxyphenylglycine, p-aminophenol, and 4-hydroxycoumarin were present at very low concentrations in both aqueous extract and 5-uRCK. Based on these results, we investigated the effect of 5-uRCK and ellagic acid on the activation of lipolysis in differentiated mature adipocytes.

First, we measured the effects of 5-uRCK and ellagic acid on glycerol 3-phosphate dehydrogenase (GPDH) activity and TG levels in mature adipocytes. GPDH plays an important role in the conversion of glycerol to TG, especially in adipocytes, and it is a marker of the late phase of adipocyte differentiation [28]. The effects of 5-uRCK and ellagic acid on GPDH activity in mature 3T3-L1 adipocytes are shown in Figure 2A,B. Both 5-uRCK (1–100 μg/mL) and ellagic acid (0.1–10 μM) significantly inhibited GPDH activity in a dose-dependent manner after incubation for 3 days. Furthermore, high doses of 100 μg/mL 5-uRCK and 10 μM ellagic acid inhibited TG content by 26.59 ± 2.61% and 25.27 ± 1.82%, respectively after incubation for 3 days, compared to the untreated control (Figure 2C,D). We assessed the lipolysis of mature adipocytes by microscopy using Oil Red O (ORO) staining and measured the lipid content. As shown in Figure 2E,I, 5-uRCK reduced the number of lipid droplets as marked by positive ORO staining during microscopy and increased the quantitative measurement of lipolysis. In accordance with these results, 5-uRCK significantly reduced lipid contents by 8.6%, 12.5%, 14.3%, 15.8%, and 19.5% at concentrations of 1, 3, 10, 30, and 100 μg/mL, respectively. Additionally, the accumulation of ORO-stained lipid droplets was visible in most of the untreated control 3T3-L1 adipocytes; however, the number of detectable stained droplets decreased in the presence of ellagic acid in mature adipocytes (Figure 2F). To investigate whether 5-uRCK or ellagic acid regulates triacylglycerol metabolism in adipocytes, differentiated 3T3-L1 cells were evaluated for lipolysis after a 24 h treatment. As depicted in Figure 2G,H, incubation with 5-uRCK or ellagic acid increased the basal level of glycerol released into the media in a dose-dependent manner after 24 h of treatment.

We next investigated in vivo the antiobesity action of 5-uRCK in an HFD-induced obese mouse model (Figure 3). Initial body weights at the beginning of the experiment were not different among the six groups. HFD-induced obese mice were obtained after 7 weeks of treatment when the weight difference was over 25% (p < 0.001). After 16 weeks of treatment, the mice fed an HFD developed 43% higher body weights than the normal-fat diet (NFD) control mice (30.69 g compared with 54.11 g). Body weight loss was not significant in 5-uRCK (100 mg/kg/day)-treated animals fed the NFD diet compared with the NFD-fed animals (Figure 3A,B). These results suggest that treatment with 5-uRCK did not induce in vivo toxicity in mice. Interestingly, both the animals fed an HFD + 5-uRCK (50 mg/kg/day) and an HFD + 5-uRCK (100 mg/kg/day) for 7 weeks showed a reduction in body weight of approximately 40% compared with animals in the HFD group (p < 0.05). The reduction in body weight was unlikely to be due to changes in food intake because 5-uRCK treatment did not significantly affect average food intake in either NFD- or HFD-fed mice (data not shown). Food intake results were also similar to our previously reported results [21,22,23,24]. As shown Figure 2C, the mortality rates of the NFD-fed group and 5-uRCK-treated group fed the NFD diet were 0% at 16 weeks. However, the mortality rate of the HFD-fed group was 22% at 14 weeks. The survival rates of the 5-uRCK 30, 5-uRCK 50, and 5-uRCK 100 groups fed the HFD diet were 89%, 100%, and 100% at 14 weeks, respectively. In addition, yo-yo dieting significantly reduced body weight gain in the 5-uRCK (100 mg/kg)-treated mice compared to untreated HFD mice (p < 0.05) (Figure 3D). Representative images of the mice are shown in Figure 3E. Mice fed an HFD were rounder and larger in size than those in the NFD group; moreover, 5-uRCK (50 and 100 mg/kg)-treated mice had a slimmer appearance than the HFD-fed mice, which might be associated with reduced fat accumulation.

The effects of dietary 5-uRCK on body weight change are shown in Figure 4. Initial body weights at the beginning of the experiment were not different among the five groups. Mice were maintained on an HFD for 3 weeks and then treated with 5-uRCK plus the HFD for another 5 weeks. In the three successive weeks of HFD feeding, the body weight gain in the HFD group was significantly higher (p < 0.05) than that in the NFD group. Furthermore, mice in the HFD group showed a significant increase (p < 0.001) in body weight compared with those in the NFD control group after 8 weeks of treatment (Figure 4A,B). However, the administration of 5-uRCK at 30, 50, or 100 mg/kg daily for 5 weeks resulted in a significant decrease in body weight (p < 0.05) compared with mice in the HFD group. The epididymal fat pad and liver weights in the HFD-fed group were higher than those in the NFD-fed control group. The 5-uRCK (100 mg/kg)-treated group had significantly (p < 0.05) reduced liver weights compared to the HFD group (Figure 4C and Figure S1a). As shown Figure 4D and Figure S1b, the average epididymal fat pad weight of the 5-uRCK 50 group (2.10 g, p < 0.05) and 5-uRCK 100 group (1.41 g, p < 0.001) was significantly lower than that of the HFD group (2.44 g). No abnormal clinical signs were observed during the experimental periods. Additionally, there were no significant differences among the HFD feeding groups in food intake (data not shown). Food intake results were also similar to those previously reported [21,22,23,24]. Figure 4E shows the morphology of the mice in different groups before sacrifice. Mice fed an HFD were rounder and larger in size than those fed an NFD. Body width was reduced in the mice treated with 5-uRCK, which might be associated with reduced fat accumulation.

At the end of the treatment, effects on plasma lipid parameters—TG, total cholesterol (TC), HDL-C, and LDL-C levels—were analyzed and are presented in Table 1. HFD-fed mice had significantly increased levels of several metabolic risk factors, including TG, total TC, and LDL-C. 5-uRCK-supplemented HFD mice showed a significant decrease in plasma TG by 10.4% (p < 0.05) at a 100 mg/kg/day dose compared with HFD-fed mice. Similarly, TC levels were significantly decreased in the 5-uRCK-treated HFD group by 10.7% (p < 0.05) at a 100 mg/kg/day dose. However, the TG and TC levels were not significantly different between the HFD group and HFD + 5-uRCK 30 or HFD + 5-uRCK 50 groups (p > 0.05). Next, we determined HDL-C and LDL-C levels in plasma. 5-uRCK treatment significantly increased HDL-C levels by 83.3% (p < 0.001), 103.6% (p < 0.001), and 92.9% (p < 0.001) at 30, 50, and 100 mg/kg/day doses, respectively. LDL-C levels were robustly increased in HFD-fed animals (p < 0.001), and 5-uRCK reduced LDL-C levels by 12.9% (p > 0.05), 13.7% (p < 0.05), and 21.4% (p < 0.01) at 30, 50, and 100 mg/kg/day doses, respectively, compared with those in the HFD diet group.

As shown in Table 1, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), amylase, uric acid (UA), and lactate dehydrogenase (LDH) were then assayed, and the HFD + 5-uRCK 100 treatment was found to significantly decrease these levels in the mice (p < 0.01, p < 0.001, p < 0.001, p < 0.001, p < 0.001 and p < 0.001, respectively). To test whether 5-uRCK could lower blood glucose levels in HFD-induced obese mice, blood glucose levels were also measured. The HFD-fed mice exhibited higher blood glucose levels than the NFD control mice, and the HFD + 5-uRCK-treated mice showed lower glucose levels than the HFD-fed mice, although the difference was not significant (p > 0.05).

As shown in Figure 5A, consuming an HFD for 3 weeks made mice obese with higher body weights than NFD-fed mice (28.08 ± 0.48 g vs. 23.87 ± 0.54 g, p < 0.05). After the induction of obesity in mice fed an HFD, ellagic acid was orally administered once per day for an additional 5 weeks. The administration of ellagic acid at 1, 2, and 4 mg/kg daily for 5 weeks resulted in a significant decrease in body weight (p > 0.05, p < 0.001 and p < 0.001, respectively) compared with mice in the HFD group (Figure 5B). Interestingly, the administration of ellagic acid (4 mg/kg) significantly reduced the body weight of HFD-fed mice from 33.17 ± 1.3 g to 31.53 ± 1.1 g (week 5 versus week 8 of treatment with ellagic acid, p < 0.001), which is almost similar to the weights of NFD-fed mice (29.27 ± 0.9 g, 8 weeks). The ellagic acid (1, 2, and 4 mg/kg)-treated group exhibited significantly (p < 0.05, p < 0.001 and p < 0.001, respectively) reduced total liver weight compared to the HFD group (Figure 5C and Figure S2a). The weights of inguinal white adipose tissue (ingWAT), epididymal white adipose tissue (eWAT), and mesenteric white adipose tissue (mWAT) were increased by the HFD (p < 0.001 for all), whereas the administration of ellagic acid to HFD-fed mice reduced ingWAT, eWAT, and mWAT weights to a similar weight as each WAT in NFD-fed mice (Figure 5D–F and Figure S2b–d). Interestingly, the relative weight of interscapular brown adipose tissue (iBAT) was increased by 1.2-fold (p < 0.001) and 1.5-fold (p < 0.001) in the ellagic acid (2 and 4 mg/kg, respectively) supplemented groups compared to that in the HFD group, whereas that in the 1 mg/kg ellagic acid-supplemented group was not different (p > 0.05) (Figure 5G and Figure S2e).

To study whether post-treatment with ellagic acid could alleviate metabolic disorders in obese mice, the mice were fed an HFD + ellagic acid diet for 8 weeks. Serum lipid profiles, including TG, TC, LDL-C, and HDL-C, of the five groups of mice are shown in Table 2. HFD feeding increased the serum levels of TG (2.30-fold), TC (1.55-fold), and LDL-C (1.43-fold) compared to NFD feeding. Ellagic acid administration markedly prevented the elevation of serum TG by 52.03% (p < 0.001), 28.53% (p < 0.001), and 52.07% (p < 0.001) (1, 2, and 4 mg/kg, respectively). TC levels were also reduced by ellagic acid treatment by 7.66% (p > 0.05), 27.77% (p < 0.001), and 27.94% (p < 0.001) (1, 2, and 4 mg/kg, respectively). Furthermore, the increased LDL-C level in the HFD group was reduced by treatment with ellagic acid by 10.92% (p > 0.05), 31.98% (p < 0.01), and 34.36% (p < 0.001) (1, 2, and 4 mg/kg, respectively). Additionally, we measured serum ALT and AST, which are clinical markers of liver damage, and our results showed that ellagic acid significantly reduced the HFD-induced increases in AST and ALT serum levels compared with those in HFD-fed mice. The ellagic acid-treated groups also had a significant reduction in CRE, amylase, and LDH levels compared with the HFD group. Moreover, the uric acid (UA) level was higher in the HFD group than in the NFD group, but these levels were not significantly different between the ellagic acid-treated group and the HFD-fed group. Notably, the administration of ellagic acid at either 1 mg/kg or 4 mg/kg significantly attenuated blood glucose levels compared with those of the HFD group.

To confirm the changes in adipocyte sizes and the number of lipid droplets in the liver, hematoxylin and eosin (H&E) staining of eWAT and liver tissue was performed, and the sizes of adipocytes and lipid droplets in the liver were analyzed. In eWAT, marked fat accumulation was observed in the HFD group (Figure 6A,B), adipocyte sizes were markedly increased (Figure 6C), and these features were inhibited in the 5-uRCK- and ellagic acid-treated groups. In all treatment groups, the relative adipocyte size was significantly decreased by treatment with 5-uRCK (50 and 100 mg/kg) and ellagic acid (1, 2 and 4 mg/kg). In the HFD group, animals exhibited hepatic lipid accumulation, and 5-uRCK and ellagic acid administration also markedly alleviated these conditions (Figure 6D).

Previous results showed that 5-uRCK and ellagic acid could decrease and inhibit body weight and adipocyte size compared with the HFD alone. Therefore, we further investigated the molecular mechanism of the lipolytic and thermogenic antiobesity effects of 5-uRCK and ellagic acid in adipocytes by protein expression analysis. The protein levels of lipolysis-associated proteins, such as ATGL, p-HSL, and PLIN1, were significantly increased in the post-treatment 5-uRCK- or ellagic acid-supplemented groups compared with those in the HFD groups (Figure 7).

PPARα, CPT1, UCP1, and PGC1α expression levels were measured to examine the effect of 5-uRCK and ellagic acid on thermogenesis. As shown in Figure 8, inguinal white adipose tissue expression of thermogenic proteins such as UCP1 and PGC1α was significantly reduced in the mice fed the HFD compared to those fed the NFD. UCP1 and PGC1α protein levels were significantly increased in the 5-uRCK- or ellagic acid-supplemented groups compared with those in the HFD groups. The expression of CPT1, an enzyme involved in mitochondrial fatty acid oxidation, was significantly increased in both the 5-uRCK and ellagic acid groups compared with the HFD group. Additionally, the protein expression of PPARα, an upstream transcription factor of CPT1, was markedly decreased in the HFD group but was increased in both the 5-uRCK and ellagic acid-post-treated groups.

The effects of dietary 5-uRCK on body weight change are shown in Figure 9. In this experiment, mice with similar initial body weights were randomly assigned into five groups (n = 8 per group). By the end of 8 weeks, the final body weight of the HFD group (42.29 ±4.03 g, p < 0.001) was significantly higher than that of the NFD group (25.50 ± 3.75 g) (Figure 9A). Pretreatment with 5-uRCK (30, 50, and 100 mg/kg) markedly inhibited weight gain in HFD-fed mice (by approximately 24.9%, 18.9%, and 30.1%, respectively) (Figure 9B). Liver weight in mice fed an HFD significantly increased by 48.7% (p < 0.01) compared to that in NFD-fed mice, and 5-uRCK (30, 50 and 100 mg/kg) inhibited liver weight by 8.70% (p > 0.05), 22.82% (p < 0.05), and 22.09% (p < 0.05), respectively (Figure 9C and Figure S3a). Epididymal adipose tissue weights from mice fed an HFD plus 5-uRCK (30, 50, and 100 mg/kg) were significantly inhibited by approximately 27.11%, 26.37%, and 31.09% (p < 0.05 for all), respectively (Figure 9D and Figure S3b). Figure 9E shows the morphology of the mice in different groups before sacrifice. Mice fed an HFD were rounder and larger in size than those fed an NFD. Body width was also reduced in the mice treated with 5-uRCK, which might be associated with inhibited fat accumulation.

As shown in Figure 10, to investigate the obesity prevention effect of ellagic acid, mice were fed an HFD with ellagic acid (1, 2, and 4 mg/kg, oral gavage daily) for 2 weeks as a pretreatment and then for 6 weeks as a cotreatment. At the end of the 8-week treatment period, HFD mice had a mean body weight gain of 41.57 ± 1.92 g, whereas NFD mice exhibited a gain of 28.42 ± 5.53 g. The mean body weight gain in HFD mice was significantly inhibited by ellagic acid (2 and 4 mg/kg) supplementation (p < 0.01 and p < 0.01, respectively) (Figure 10A,B). The effects of ellagic acid supplementation on fat accumulation in the liver were investigated. Furthermore, to investigate whether the ellagic acid-induced body weight reduction was associated with the decrease in various fat pads, the pads were dissected and weighed. The results demonstrated that these organs were significantly heavier in HFD mice than in NFD mice and that ellagic acid treatment significantly inhibited fat deposition in these organs compared with those in HFD-fed mice (Figure 10C–F and Figure S4a–d). All WAT weight increases were significantly inhibited by ellagic acid. Interestingly, the relative weight of iBAT was increased by 51.6% (p < 0.05) and 50.7% (p < 0.01) in the ellagic acid (2 and 4 mg/kg, respectively)-supplemented groups compared to that in the HFD group, whereas the 1 mg/kg ellagic acid-supplemented group was not different (p > 0.05) (Figure 10G and Figure S4e).

HFD-fed mice exhibited significantly higher serum levels of TG (55.15%, p < 0.001), TC (87.67%, p < 0.001), and LDL-C (79.99%, p < 0.001) and significantly lower levels of HDL-C (47.66%, p < 0.001) than mice in the NFD group. 5-uRCK significantly inhibited TG (30, 50, and 100 mg/kg), TC (50 and 100 mg/kg), and LDL-C (50 and 100 mg/kg) increases and increased HDL-C levels (30, 50, and 100 mg/kg) compared with HFD alone (Table 3). Treatment with 5-uRCK significantly and dose-dependently inhibited the increases in the levels of TG, TC, and LDL-C, and at the highest dose examined (100 mg/kg), these lipid profile increases were inhibited by 31.1%, 20.4%, and 23.1%, respectively, compared with those in HFD-fed mice. Moreover, 5-uRCK (100 mg/kg) increased HDL-C by 98.2% compared with that in HFD-fed mice. Furthermore, ellagic acid treatment also dose-dependently inhibited serum TG, TC, and LDL-C levels and increased HDL-C levels compared with those in HFD-fed mice (Table 4). Ellagic acid at a dose of 4 mg/kg decreased the mean TG, TC, and LDL-C levels by 31.8% (p < 0.001), 19.0% (p < 0.001) and 34.7% (p < 0.001), respectively, compared with an HFD alone. Moreover, ellagic acid (4 mg/kg) increased HDL-C by 71.9% (p < 0.001) compared with an HFD alone.

As shown in Table 3, AST, ALT, CRE, amylase, UA, and LDH were assayed, and the HFD + 5-uRCK 100 treatment significantly inhibited these in the mice (p < 0.001, p < 0.001, p < 0.001, p < 0.05, p < 0.001 and p < 0.001, respectively). To test whether 5-uRCK could lower blood glucose levels in HFD-induced obese mice, blood glucose levels were also measured. The HFD-fed mice exhibited higher blood glucose levels than the NFD control mice, while the HFD + 5-uRCK-treated groups showed lower glucose levels than the HFD-fed mice, with no significant difference (p > 0.05). In addition, as shown in Table 4, ellagic acid significantly reduced the levels of AST, ALT, CRE, amylase, UA, and LDH in a similar manner to 5-uRCK. Interestingly, blood glucose levels were significantly reduced in all groups treated with ellagic acid at 1, 2 and 4 mg/kg (p < 0.05, p < 0.05 and p < 0.001, respectively).

As shown in Figure 11, H&E staining revealed enlarged or hypertrophic eWAT in the HFD group and smaller eWAT sizes in 5-uRCK- or ellagic acid-supplemented mice (Figure 11C). The adipocyte area of the HFD group was approximately 12,000 μm², which was significantly greater than that of the NFD group. Intake of 30–100 mg/kg 5-uRCK with an HFD significantly inhibited adipocyte area to approximately 8400, 2800, and 2300 μm². The 2 mg/kg ellagic acid inhibited the area to 5100 μm², and 4 mg/kg ellagic acid significantly inhibited it to 3300 μm², suggesting a dose-dependent effect of 5-uRCK and ellagic acid on adipocyte size. In addition, the histological study revealed enlarged hepatocytes, as evidenced by excessive vacuolation in the HFD group compared with the NFD group. As presented in Figure 11D, 5-uRCK or ellagic acid markedly reduced the lipid droplet number in the liver tissues of HFD mice.

The protein levels of lipolysis-associated proteins, such as ATGL, p-HSL, and PLIN1, were significantly increased in both the pretreatment 5-uRCK- or ellagic acid-supplemented groups compared with those in the HFD groups (Figure 12).

As shown in Figure 13, inguinal white adipose tissue expression of thermogenic proteins such as UCP1 and PGC1α was significantly reduced in the mice fed the HFD compared to those fed the NFD. UCP1 and PGC1α protein levels were significantly increased in the 5-uRCK- or ellagic acid-supplemented groups compared with those in the HFD groups. The expression of CPT1 significantly increased in both the 5-uRCK and ellagic acid groups compared with the HFD group. Additionally, the protein expression of PPARα was markedly increased in both the 5-uRCK and ellagic acid-pretreated groups.

3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), Oil Red O, dimethyl sulfoxide (DMSO), and ellagic acid (EA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin (BSA), and fetal bovine serum (FBS) were purchased from Invitrogen, Inc. (Grand Island, NY, USA). All other chemicals were of analytical reagent grade.

The unripe R. coreanus (uRCK, specimen voucher number: BT-URCK001) fruits used in this study were collected (June 2018) in Gwangyang County (Jeollanam-do, Korea) and authenticated by Dr. Kim at B&Tech, Gwangju, South Korea. uRCK (2.5 kg) was extracted using 20 volumes of 5% ethanol at 100 for 4 h, as described in our previous study [21,22,23,24,25,62,69]. The extracted solution was then filtered, concentrated with an evaporator under vacuum, and freeze-dried. The dry matter content of the lyophilized samples was determined by drying at 105 °C to a constant mass. Normally, 20.4 g of dried powder could be obtained from 100 g of unripe R. coreanus.

The dried 5-uRCK (30 g) was suspended in distilled water and successively divided with n-hexane (3 × 500 mL), chloroform (CHCl₃, 3 × 500 mL), ethyl acetate (EtOAc, 3 × 500 mL), and n-butanol (BuOH, 3 × 500 mL). The ethyl acetate fraction of 5-uRCK (18 g) was loaded onto a silica gel column (length: 32 cm, diameter: 2 cm, Merck, Darmstadt, Germany) and eluted with a stepwise gradient of chloroform, ethyl acetate, and methanol. The collected active subfractions were further separated by preparative thin layer chromatography (TLC), since the active isolate was not pure. It was again purified on an LH-20 column (length: 20 cm, diameter: 1.5 cm) using methanol as the eluant. The isolated compound was pure by high-performance liquid chromatography (HPLC). The amounts of ellagic acid were analyzed with HPLC and compared with a standard preparation of ellagic acid produced with our previously reported standard method [21,22,23,24,25,62].

The 3T3-L1 preadipocytes were purchased from ATCC (American Type Culture Collection, CL-173, Manassas, VA, USA) and cultured in DMEM/high glucose supplemented with 10% newborn calf serum and 1% penicillin and streptomycin, which was defined as complete media, in a CO₂ incubator at 37 °C. Preadipocytes were cultured and differentiated into adipocytes. Briefly, preadipocytes were cultured in complete medium. Two or three days after reaching confluence, cells were stimulated with differentiation medium (MDI) prepared by adding 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methyxanthine (IBMX), and 10 μg/mL insulin to complete medium (day 0). After two days (day 2), the cells were stimulated with 10 μg/mL insulin in complete medium. After three days (day 5), the differentiation media were replaced with complete media. The cell culture media were changed every 2 days until full differentiation, that is, up to 9 days (day 14). To investigate the effect of 5-uRCK or ellagic acid on lipolysis in mature 3T3-L1 adipocytes, cells were maintained in the presence or absence of 5-uRCK or ellagic acid from day 14 to day 17 (72 h) in six-well plates.

For lipid accumulation, Oil Red O staining was performed for 3T3-L1 cell lines. Mature adipocytes were incubated in complete media, with or without 5-uRCK or ellagic acid, for 72 h, and the cells were fixed with 4% formaldehyde for 20 min. Then, cells were washed with 60% isopropanol and fully dried. Oil Red O working solution was added, and cells were maintained at 30 min at room temperature. Mature fat cells were stained with Oil Red O solution. Excess stain was removed by washing thoroughly with distilled water. Fully differentiated mature fat cells were photographed using an inverted microscope (Nikon Instrument, Melville, NJ, USA). Differentiation was monitored using microscopy and quantified via elution with 100% isopropanol. Optical density (OD) measurements were obtained at 490 nm. GPDH activity was measured with a GPDH activity assay kit (Takara Bio Inc., Shiga, Japan) in accordance with the manufacturer’s instructions. Total triglyceride content in the mature adipocytes was measured; 100% isopropanol was added to mature adipocytes and incubated at room temperature for 10 min followed by measurement of absorbance at 630 nm using a TG assay kit (Asan pharmaceuticals, Seoul, Korea). Values for TG levels were expressed as a percentage of the control.

The matured adipocytes were gently washed once with phosphate-buffered saline (PBS) and then incubated with DMEM (no phenol red and FBS) containing or without 5-uRCK or ellagic acid for 24 h. The culture medium of samples was collected, and glycerol levels in the media were assayed using a glycerol kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s instructions.

Specific pathogen-free (SPF) grade healthy male four-week-old C57BL/6 mice weighing 18–21 g each were purchased from Central Lab Animal, Inc. (Seoul, Republic of Korea). Animals were maintained at a constant room temperature of 22 ± 2 °C with a humidity level of 50 ± 5% and with free access to water and food under a 12:12 h light:dark cycle (lights on at 8:00 am). The animals were acclimatized for 4 days before the beginning of the experiments. All efforts were made to minimize animal suffering and to reduce the number of animals used. The experiment was conducted according to the International Guidelines for the Care and Use of Laboratory Animals [70] and was approved by the institutional animal care and use committee (IACUC) of the Bioresources and Technology (B&Tech, Gwangju, Korea) Co., Ltd., Republic of Korea (Approval number: BT-004-2018, 9 July 2018). When the experiment began, all mice were anesthetized with isoflurane and then sacrificed by cervical dislocation in accordance with the IACUC guidelines.

The mice were randomly assigned to each treatment group consisting of 8–9 mice each (Table 5). The normal-fat diets consisted of 10% kcal from fat (Research Diets, D12450B, New Brunswick, NJ, USA). The HFD consisted of 60% kcal from fat (Research Diets, D12492, New Brunswick, NJ, USA). 5-uRCK (30, 50, and 100 mg/kg) or ellagic acid (1, 2, and 4 mg/kg) of 0.2 mL, dissolved in physiological saline solution, was orally administered using a stainless oral sonde (Jungdo-BNP, Seoul, South Korea), and the control rats received the same volume of saline solution. This dosage was calculated from an amount of 600 mg of 5-uRCK extract per day per 60 kg of human body weight. Food and water intake were measured every day, and body weight was measured once every two days. The feed efficiency ratio (FER) was calculated as follows: FER = G × F⁻¹, where G is the weight gain (g) and F is the consumption (g) of dry matter from the feed [21,22,23,24].

At the end of the experimental period, the mice were fasted for 12 h prior to sacrifice. Blood samples were collected from the orbital venous plexus of mouse eyes and centrifuged at 1000× g and 4 °C for 15 min. Plasma was stored at −70 °C prior to the experiments. The concentrations of serum TC, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose were measured using an Alere cholesterol LDX^® system (Cholestech LDX, Hayward, CA, USA). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assays were performed with a kit according to the manufacturer’s instructions (Asan Pharm, Seoul, Korea). CRE, amylase, UA, and LDH assays were carried out using commercial kits in an automated biochemical analyzer (DRI-CHEM NX500i, FUGI-FILM, Tokyo, Japan). After blood collection, the adipose tissue (epididymal fat, perineal fat) and liver were removed from the mice and immediately weighed.

The epididymal adipose tissue was fixed in 4% formaldehyde and stored at −80 °C. Six-micron-thick sections of frozen adipose tissues were prepared using a cryomicrotome and stained with H&E. The liver was collected and fixed in PBS containing 4% formalin and embedded in paraffin, and five-micron-thick sections were stained with H&E. The images were acquired by light microscopy (Olympus BX51, Tokyo, Japan), and we analyzed the images with MetaMorph^® microscopy automation and image analysis software (Molecular Devices, Sunnyvale, CA, USA). The average adipocyte size of the fat pad was quantified by capturing four independent fields per blinded slice using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and expressed in μm²/adipocyte.

Samples of epididymal or inguinal white adipose tissues were washed three times with cold PBS before being lysed in radioimmunoprecipitation (RIPA) lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 1% NP-40; 0.1% sodium deoxycholate, 0.2% SDS, 150 mmol/L NaCl, and 1 mmol/L EDTA) supplemented with 1× protease and phosphatase inhibitor cocktail (Thermo, Fremont, CA, USA) on ice. The separated proteins were transferred onto a nitrocellulose membrane. The anti-ATGL (1:100, #2138), anti-HSL (1:200, #4107), anti-p-HSL (1:200. Ser563, #4139), anti-perilipin-1 (1:100, #3467), anti-UCP1 (1:100, #14670), anti-CPT1 (1:100), PGC1α (1:100, #4259), and anti-β-actin (1:3000, #4967) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The monoclonal anti-PPARα antibody (1:200, ab191226) and secondary antibodies (1:10,000) were obtained from Abcam (Cambridge, MA, USA). Immunoreactive protein bands were visualized using a ChemiDoc XRS+ System (Bio-Rad) and quantified with Gel Pro Analyzer software (Silk Scientific, Inc., Orem, UT, USA). The internal control, β-actin, was used to normalize differences due to loading variations.

Data are presented as the mean ± standard deviation (SD) or standard error (SE) from three independent experiments with replication. Data were statistically evaluated using Student’s t-test or one-way analysis of variance (ANOVA) with GraphPad Prism 5 version 5.01 for Windows (GraphPad, Inc., San Diego, CA, USA) software programs. Statistical significance was indicated when p < 0.05.