All the experimental procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (Permit Number: KIACUC-1700114) and according to the Laboratory Animal Act of the Ministry of Food and Drug Safety.

Thirty-six male C57BL/6J mice were purchased from Samtako Bio Korea (C57BL/6NTac, Samtako Inc., Osan, Korea) at 4 weeks of age and housed in a specific pathogen-free facility. They were individually housed in a cage where the temperature was maintained at 23 ± 3℃ with relative humidity of 50 ± 10%, in a 12 h light/dark cycle.

All the mice consumed a commercial diet and tap water ad libitum for 1 week prior to their allocation to one of the three weight-matched groups; the Chow group (low-fat diet, provided 4,057 ㎉% with 10% fat, 20% proteins and 70% carbohydrates, D12450B, Research Diets, Inc., New Brunswick, NJ, USA) an HFD group (high-fat diet, provided 4,057 ㎉% with 45% fat, 20% proteins and 35% carbohydrates, D12451, Research Diets Inc., New Brunswick, NJ, USA), and HFD with RPA group (HFD + RPA).

The RPA contained 125.57 ㎎ total anthocyanins containing 118.90 ㎎ C3G and 6.67 ㎎ of C3R per gram. RPA (5,000 ㎎/㎏/day) was administered orally every day for 12 weeks. After 12 weeks, the mice were sacrificed by decapitation. Blood samples, the heart, the liver, the kidney, and adipose tissue were collected, weighed and stored at -80℃. Body weight and food consumption measurements were started in the first week of the study (5 weeks of age) and continued weekly for the entire period of the experiment.

Food consumption was determined for each of the three groups by weighing the total amount of food given at the start of each week and then subtracting the amount of food remaining at the end of the week. The average food consumed per mouse was then calculated by dividing by the number of the mice.

The experimental diets were fed for 12 weeks. On the last day of the experiment and after 12 h of fasting, the mice were euthanized using CO₂ gas and dissected. Blood was collected via cardiac puncture into heparinized tubes using a syringe and left at room temperature to coagulate for 20 min before centrifugation at 3,000 × g at 5℃ for 15 min.

The resultant serum was stored at -70℃ until analysis. The liver, heart, kidney, spleen, testes, and epididymis [epididymal white adipose tissue (WAT) and epididymal brown adipose tissue (BAT)] were harvested. Each organ was rinsed in physiological saline, weighed, and stored in a freezer at -70℃ for later use.

Blood glucose levels were determined using a chemistry analyzer (Hitachi 7600, Hitachi Co., Tokyo, Japan). Serum total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-CH) levels were determined using commercial enzymatic kits and an automatic biochemistry analyzer (Roche Cobas 8000 modular analyzer Series C702, Mannheim, Germany). Low-density lipoprotein cholesterol (LDL-CH) was calculated using the Friedewald formula (TC - HDL - TG/5). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in plasma were determined using a commercially available kit purchased from Asan Pharmaceutical (AM101-K, Seoul, Korea).

Serum insulin, leptin and adiponectin levels were analyzed by immunoassay using a rat/mouse ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturer’s protocols. Homeostatic model assessment insulin resistance (HOMA-IR) was calculated using the following formula:

After the mice are sacrificed, the abdominal tissue is separated and made into small pieces. For the oil red O staining, hepatic tissues were frozen in liquid nitrogen, sliced and stained with oil red O solution (0.5 g/100 ㎖, dissolved in isopropanol). For the hematoxylin and eosin (H&E) staining, epididymal adipose tissues were frixed in 10% formaldehyde solution neutralized to pH 7.4, embedded in paraffin, sliced and stained. All the section images were captured with a light microscope (Olympus, Tokyo, Japan).

The liver samples from each mouse were homogenized in PBS, and total lipids were determined according to a previously described method (Folch et al., 1957).

The liver homogenate was centrifuged at 1,900 × g for 20 min at 4℃ and the supernatant was stored on ice until analysis. The concentrations of liver triglycerides and total cholesterol were estimated using the same enzymatic kits and analyzer for serum analysis.

Total RNA from the liver and WAT was extracted with Trizol (Invitrogen Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Single-stranded cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzbeng, Germany). Quantitative PCR was performed using SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan).

The 20 ㎕ reaction mixture was prepared as follows: 10 ㎕ SYBR Green Quantitative PCR SuperMix-UDG (Invitrogen Technologies, Carlsbad, CA,. USA), 0.4 ㎕ of forward primer (10 μM), 0.4 ㎕ of reverse primer (10.0 μM), and cDNA (2.0 ㎕). The real-time PCR conditions were as follows: 95℃ for 10 min followed by 45 cycles at 95℃ for 15 s, 60℃ for 5 s; 72℃ for 15 s. The primer sequences used in the experiments are listed in Table 1.

All results were obtained from at least three independent experiments. The liver expression of PPARγ, FAS, ACO, CPT 1 and the WAT expression of IL-1β, IL-6 and TNFα were examined and normalized using β-actin as an internal control.

The sample groups were statistically analyzed using SPSS 19.0 statistical software (Statistical Package for the Social Sciences, ver. 19.0, SPSS Inc., Chicago, IL, USA). Means ± standard error for each group were calculated. Significant differences among groups were examined by post-hoc Duncan’s Multiple Range Tests (DMRT, p < 0.05).

All mice treated with RPA for 12 weeks were healthy. The initial body weight of mice averaged 18.12 g, which was not significantly different among the three groups.

The effects of RPA on body weight are shown in Table 2. After 12 weeks, the body weight gain of high-fat diet (HFD) group (35.76 ± 3.54 g) was significantly higher than that of the Chow group (10.44 ± 1.12 g). The intake of RPA reduced body weight in the HFD-fed mice by 37.24%, but their body weight was still higher than that of the mice in the Chow group (Table 2 and Fig. 1A).

Fig. 1.  Representative macroscopic pictures of male C57BL/6J mice from the different groups at the end of the experiment.

A; C57BL/6J mice, B; liver, C; heart, D; epididymal white adipose tissue. First panel; reference Chow (low-fat diet) diet, second panel; HFD (high-fat diet), third panel; HFD + RPA (HFD plus 5,000 ㎎/㎏ purified mulberry anthocyanin using PB-600 macroporous resin).

Furthermore, there were no statistically significant differences in body length among the three groups (Table 2 and Fig 1A). The food efficacy ratio (FER) of the HFD + RPA group (7.22 ± 1.26%) was significantly lower than that of the HFD group (19.65 ± 0.87%) (p < 0.01), and there were no significant changes in the daily food intake between the other groups after treatment (Table 2). This results suggested that anti-obese property of anthocyanins relies on its ability to control food consumption and energy metabolism, which was witnessed by the increase in energy expenditure, suppression of weight gain.

Skates et al. (2018) reported that the consumption of anthocyanin rich berry improved body composition via enhancing energy expenditure, amended mitochondrial respiration and reduced metabolic stress in the high-fat-diet-fed C57BL/6 mouse.

The weights of the heart, liver, kidney and interscapular brown adipose tissue were lower in HFD-fed mice than in low-fat fed mice (Chow group), and the HFD + RPA group also showed a relatively higher tissue weights than the HFD group (Table 2). The weight of white adipose tissue (WAT) was much higher in the HFD group than in the Chow group, but it was decreased after RPA administration.

Supplementation with anthocyanin-rich Norton grape pomace extract (250 ㎎/㎏ of body weight/day) for 12 weeks exhibited anti-inflammatory effects, there was no significant change in the body weight of experimental obese mice whose high-fat diet was supplemented with grape extract (Hogan et al., 2010). However, the supplementation with blueberry and mulberry (Morus alba L.) juice or mulberry anthocyanins (40 or 200 ㎎/㎏) for 12 weeks significantly suppressed body weight gain and reduced insulin resistance and lipid accumulation in HFD-fed mice (Wu et al., 2013).

The results showed that anthocyanin-rich berries may more effectively hinder diet-induced obesity than anthocanins from other sources. In addition, RPA regulates lipogenesis and lipolysis in the high-fat diet induced experimental obese C57BL/6J model, and RPA might be considered as a supplementary therapeutic agent to manage obesity.

Mice in the HFD group showed elevated serum glucose, triglyceride, and total cholesterol levels than Chow group (Table 2). RPA decreased triglyceride and total cholesterol levels, while it significantly reduced serum glucose levels. The HFD increased the LDL-C, ALT and AST levels, whereas RPA supplementation decreased their levels to those of the Chow group but did not affect HDL-C (Table 2).

Obesity is most likely to cause hyperlipidemia which is considered a leading cardiovascular risk. The hallmark of dyslipidemia in obesity is hypertriglyceridemia combined with the preponderance of high LDL and low HDL cholesterol (Klop et al., 2013).

Bhaswant et al. (2019) investigated that Queen Garnet (QG) plum juices containing small amount anthocyanins (250 ㎖ juice per day; 102 ㎎·C3G equivalents of total anthocyanins) significantly reduced blood pressure, fasting plasma LDL, glucose, insulin, C-peptide, GLP-1 (glucagon-like peptide-1) and leptin levels, whereas, there were no changes in TC, TG, ALT, AST, PAI-1 (plasminogen activator inhibitor-1), and creatinine in QG juice interventions.

In our previous study, we assessed the effect of a low dosage of RPA on a 3T3-L1 preadipocyte differentiation model and a process for the industrial preparation of mulberry anthocyanins using PB-600 macroporous resin (Kim et al., 2021). Mulberry anthocyanin purified using PB-600 macroporous resin (RPA) contained 152.37 ㎎/g C3G equivalents of total anthocyanins.

In this study, the amount of anthocyanin used in RPA was 5,000 ㎎/㎏/day (761.85 ㎎·C3G equivalent/g), which was 7.5 times higher than those in Queen Garnet plum juices’s study by Bhaswant et al. (2019). It suggest that high doses of C3G are required to reduce blood pressure and risk factors associated with metabolic disorders, and reverse obesity reversal.

Serum leptin, insulin, adiponectin and insulin resistance index were examined (Fig. 2). Serum leptin was elevated in mice fed HFD compared to the Chow group (4.1 ± 0.5). HFD-fed obese mice showed significantly higher levels of serum leptin, but the HFD + RPA group (5.1 ± 0.3) showed 75.8% lower levels of serum leptin than the HFD group (7.8 ± 0.2).

Fig. 2.  Serum insulin levels, leptin levels, HOMA-IR, and adiponectin in mice.

Chow; low-fat fed mice group, provided 4,057 ㎉% with 10% fat, HFD; high-fat fed mice group, provided 4,057 ㎉% with 45% fat, HFD + RPA; high-fat fed mice group + RPA (5,000 ㎎/㎏/day, 118.90 ㎎ C3G and 6.67 ㎎ of C3R per gram) were orally administered every day for 12 weeks. Values are mean ± SEM (n = 12). *Means within a column followed by the same letter are not significantly different based on the Duncan`s Multiple Range Test (DMRT, p < 0.05).

Leptin, the product of an obesity-related gene, is secreted by adipose tissues and has an important function in lipid metabolism (Maratos-Flier, 2008). It regulates food intake and energy expenditure (Rosenbaum and Leibel, 1999), and tis levels are correlated with obesity, especially visceral fat accumulation (Kershaw and Flier, 2004).

In this study, treatment of HFD-fed obese mice with RPA reduced leptin levels. The administration of mulberry anthocyanin containing C3G might have improved leptin resistance in obese mice.

HFD-fed obese mice showed significantly higher levels of insulin and HOMA-IR than the Chow group mice. Whereas there was no statistically significant decrease in insulin levels in the HFD + RPA group, the lower plasma glucose (Table 2) and insulin levels resulted in markedly lower insulin resistance index (HOMA-IR) values in the HFD + RPA group as compared with the HFD group, 56.25% lower (Fig. 2, p < 0.05).

Loss of body weight in obese patients and an aninal model has been associated with improvement of HOMA-IR indicating insulin resistance (Bogardus et al., 1984), decreased glucose production (Henry et al., 1986), and increased insulin secretion (Gumbiner et al., 1990).

The adiponectin concentration was higher in the Chow group (20.10 ± 2.30 ㎎/ℓ) than in the HFD group (5.80 ± 1.20 ㎎/ℓ). RPA administration elevated adiponectin levels in HFD-fed mice (20.50 ± 2.60 ㎎/ℓ).

Adiponectin is an adipocyte secretory protein hormone that modulates metabolic processes, such as fatty acid oxidation and glucose metabolism (Arçri et al., 2009). Circulating levels of adiponectin are decreased in obese subjects. Increased concentrations of adiponectin are related to a reduction in body weight in obese animals (Gregor and Hotamisligil, 2011; Siegrist et al., 2013). This study showed that RPA potentially induces fatty acid oxidation and reduces serum glucose levels with an increased adiponectin concentration.

Dietary supplementation with mulberry anthocyanin containing C3G suppressed body weight gain, fat accumulation, and hyperlipidemia, and improved glucose homeostasis, insulin resistance, and leptin resistance.

Fig. 3 shows the Oil Red O staining of the liver and H&E staining of epididymal adipose tissue. Compared with the Chow group, mice fed with HFD showed a notable increase in fat vacuole and intense lipid accumulation in the liver (Fig. 3B). In contrast, RPA administration resulted in a dramatic decrease in fat vaculoles (both in size and number) relative to HFD, and this decrease significantly alleviated lipid accumulation in HFD-fed mice.

Fig. 3.  Morpholoogy changes in the liver (A - C) and epididymal adipose tissue (D - F) for the male C57BL/6J mice.

A - C; Oil Red O was used to stain livers sections of mice, D - H; H&E stained epididymal adipose tissue. A and D; Chow (low-fat diet mice group provided 4,057 ㎉% with 10% fat), B and E; HFD (high-fat diet mice group, provided 4,057 ㎉% with 45% fat), C and F; high-fat diet mice group + RPA (5,000 ㎎/㎏/day, 118.90 ㎎ C3G and 6.67 ㎎ of C3R per gram) administered orally every day for 12 weeks. Scale bar; 5 ㎛.

In this study, activity of the liver enzyme ALT and AST, as an indicator of liver damage, revealed lower ALT and AST levels in the HFD + RPA group than in the HFD group (Table 2). These results support the H&E staining results of the liver, which revealed that the proportion of normal cells in the group supplemented with RPA increased compared to the HFD group, suggesting that RPA has hepatoprotective effects.

Fig. 3D - 3F shows the histology of epididymal white adipose tissue of mice. Mice fed with HFD showed hypertrophy of adipocytes in the adipose tissue (Fig. 3E). The phenotype of adipocytes was attenuated when the HFD-fed mice were treated with RPA (Fig. 3F). The epididymal fat and abdominal visceral fat of the RPA-treated group decreased compared with the HFD group.

The mRNA expression levels of PPARγ, FAS, ACO and CPT 1 were determined in the liver tissue (Fig. 5A). Quantitative real-time PCR analysis was also performed to evaluate the expression of IL-1β, IL-6 and TNFα in epididymal white adipose tissue (Fig. 5B).

Compared to the Chow group, mice fed with HFD showed upregulation of PPARγ, FAS, IL-6, and TNFα genes, and downregulation of the CPT 1 gene. RPA markedly reduced the expression levels of PPARγ, FAS, IL-6 and TNFα compared to the HFD control, while increased CPT 1 expression levels.

As mentioned above, HFD-fed obese mice showed high lipid accumulation in the liver (Fig. 4A). HFD significantly increased triglyceride levels in the liver (Fig. 4B), but RPA reversed this effect. RPA consumption may regulate lipid metabolism by suppressing fatty acid synthesis-related genes (PPARγ and FAS) and inducing the expression of β-oxidation-related genes (CPT 1).

Fig. 4.  Hepatic contents of total lipids, triacylglycerol, and cholesterol.

A; liver lipids, B; liver triglycerides, C; liver cholesterol, Chow; low-fat fed mice group, provided 4,057 ㎉% with 10% fat, HFD; high-fat fed mice group, provided 4,057 ㎉% with 45% fat, HFD + RPA; high-fat fed mice group + RPA (5,000 ㎎/㎏/day, 118.90 ㎎ C3G and 6.67 ㎎ of C3R per gram) administered orally, every day for 12 weeks. Values are mean ± SEM (n = 12). *Means within a column followed by the same letter are not significantly different based on the Duncan`s Multiple Range Test (DMRT, p < 0.05).

PPARγ regulates lipid and glucose metabolism, is essential for adipocyte proliferation and differentiation and plays an important role in enhancing insulin sensitivity by promoting adiponectin expression in various peripheral tissues (Yanai and Yoshida, 2019). In addition, it suppresses the expression of inflammatory cytokines, such as tumor necrosis factor-α (TNFα) in adipocytes (Mirza et al., 2019). Adiponectin inhibits hepatic glucose production, and reduces insulin resistance by increasing glucose uptake and fatty acid oxidation in muscles (Ghadge et al., 2018).

CPT-1 mediates transport of free fatty acids (FFAs) into the mitochondria and regulates lipid metabolism by promoting β-oxidation of FFAs (Gross et al., 2017). Increased FFA levels due to lipid metabolic disorders are known to induce inflammation associated with insulin resistance, reduce glucose uptake, increase hepatic glucose production, impair insulin signaling pathways, and cause adipokine secretion disorders (Morigny et al., 2016). It is suggest that the RPA can increases PPAR-expression, which, in turn, enhances insulin sensitivity.

The effect of purified C3G on molecular pathways in adipose tissue have also been investigated in vivo by Choi et al. (2016). Db/db mice were fed C3G dissolved in drinking water (1 ㎎/㎖, 22 ㎎/㎖, or 1,320 ㎎ of C3G for a 60 ㎏ adult) for 16 weeks. After 16 weeks, the liver, and epididymal white adipose tissue also weighed significantly less in the C3G group compared to the control group, C3G-treated mice consumed approximately 30% more oxygen (higher energy expenditure) despite no difference in energy intake or physical activity.

Obesity is associated with a state of chronic low-grade systemic inflammation, which increases the production of obesity-related inflammatory cytokines, such as IL-1β, IL-6, TNFα, and leptin, and decreases anti-inflammatory cytokine levels, such as adiponectin (Samuel Wu et al., 2013; Wang et al., 2013). Inflammatory cytokines, such as TNFα and IL-6, impair insulin signaling pathways by targeting the downstream components of the insulin signaling pathway in adipose tissue. TNFα and IL-6 interfere with insulin signaling and induce lipolysis, generating circulation lipids and inducing insulin resistance (Akash et al., 2018). In the present study, mice that were supplemented with RPA showed lower TNFα mRNA expression than mice fed with HFD. We found that HFD-fed mice were under the pathologic condition of inflammation associated with obesity, as evidenced by high levels of IL-6, TNFα and leptin. Our results further indicated that RPA exerted a potentially anti-inflammatory effect (Fig. 2A and Fig. 5B)

Fig. 5.  Effects of purified mulberry anthocyanins using PB-600 macroporous resin (RPA) on mRNA expressions of PPAR-γ, FAS, ACO, CPT 1, IL-1β, IL-6 and TNFα by quantitative real-time polymerase reaction in C57BL/6J mice fed a high-fat diet for 12 weeks.

The intensity of the bands was quantified by densitometric analysis and normalized with the corresponding levels of β-actin. Chow; low-fat fed mice group, provided 4,057 ㎉% with 10% fat, HFD; high-fat fed mice group, provided 4,057 ㎉% with 45% fat, HFD + RPA; high-fat fed mice group + RPA (5,000 ㎎/㎏/day, 118.90 ㎎ C3G and 6.67 ㎎ of C3R per gram) administered orally, every day for 12 weeks. PPAR-γ; peroxisome proliferator-activated receptor, FAS; fatty acid synthase, ACO; acyl-CoA oxidase, CPT 1; carnitine palmitoyl transferase, IL-1β; iterleukin-1β, IL-6; iterleukin-6, TNFα; tumor necrosis factor α. Values are means ± SEM (n = 12. *Means within a column followed by the same letter are not significantly different based on the Duncan's Multiple Range Test (DMRT, p < 0.05).

Given these findings, we confirmed that RPA was effective in improving insulin sensitivity by promoting PPARγ expression and, reducing inflammation by decreasing TNFα expression. Thus, RPA is considered effective in alleviating the interruption of insulin signaling pathways, inhibiting lipolysis, and improving insulin resistance.

Therefore, these results suggest that RPA has positive antiobesity, and anti-inflammatory effects and could reduce obesity, and insulin resistance. In addition, the consumption of purified anthocyanin, is a better option to ensure the intake of health-beneficial high amounts rather than the fruit, fruit juice, or extract itself.

Though the preclinical studies proved the beneficial effects of purified mulberry anthocyanins, do not have an established treatment procedure to prevent or manage the over-weight condition and its comorbidities.

As mentioned earlier, anti-obese property of anthocyanins relies on its ability to control food consumption and energy metabolism, and improve inflammatory response, insulin resistance, and glucose metabolism, etc.

We are urged to discover the practical application of dietary purified mulberry anthocyanins for the treatment of obesity. Thus, further studies on the optimum dose, duration, and mode of supplementation of anthocyanins are required to develop anthocyanins-based treatment procedures.