Anti-Obesity Effects of a Prunus persica and Nelumbo nucifera Mixture in Mice Fed a High-Fat Diet

Prunus persica and Nelumbo nucifera are major crops cultivated worldwide. In East Asia, both P. persica flowers and N. nucifera leaves are traditionally used for therapeutic purposes and consumed as teas for weight loss. Herein, we investigated the anti-obesity effects of an herbal extract mixture of P. persica and N. nucifera (HT077) and the underlying mechanism using a high-fat diet (HFD)-induced obesity model. Male C57BL/6 mice were fed a normal diet, HFD, HFD containing 0.02% orlistat (positive control), or HFD containing 0.1, 0.2, or 0.4% HT077 for 12 weeks. HT077 significantly reduced final body weights, weight gain, abdominal fat weights, liver weights, and hepatic levels of triglycerides and total cholesterol. HT077 also lowered glucose, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and leptin levels and increased AST/ALT and adiponectin/leptin ratios and adiponectin levels. Real-time polymerase chain reaction analysis showed that HT077 decreased the expression of lipogenic genes and increased the expression of fatty acid oxidation-related genes in adipose tissue. Our results indicate that HT077 exerts anti-obesity effects and prevents the development of obesity-related metabolic disorders. These beneficial effects might be partially attributed to ameliorating adipokine imbalances and regulating lipid synthesis and fatty acid oxidation in adipose tissue.


Introduction
The steady increase in overweight and obese individuals has become a serious health problem worldwide. Currently, it is estimated that nearly one-third of the world's population is overweight or obese [1]. Obese people are at a high risk for many serious diseases, including diabetes, cardiovascular diseases, nonalcoholic fatty liver disease (NAFLD), osteoarthritis, and cancers [2]. Obesity and related diseases not only damage a person's health and quality of life but also have become a socioeconomic burden. For the management of such conditions, lifestyle interventions, including a low-calorie diet, physical activity, and behavior therapy, are ideal, but motivation and adherence are not easy [3]. Pharmacological interventions for long-term use include orlistat, lorcaserin, liraglutide, phentermine-topiramate, and naltrexone-bupropion. These drugs are approved for moderately or severely obese patients with a body mass index (BMI) ≥30 or BMI ≥27 with comorbidities and have a high incidence of side effects [4]. Considering the limitations of conventional medication and the complex nature of the pathogenesis of obesity, more safe and effective novel approaches are required.

Statistical Analysis
Values are expressed as the mean ± standard deviation (SD). One-way analysis of variance with Dunnett's post hoc test was performed to compare more than two groups, whereas independent t-tests were performed to compare two groups. All analyses were performed using Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was set at p < 0.05.

Effects of HT077 on Body Weight and Food Consumption
The body weights of animals in the HFD control group began to increase significantly compared with those in the ND group at week 4, and the difference between the two groups grew until the end of the study period ( Figure 1a). Supplementation with 0.2% HT077 significantly reduced body weight at 4-8 weeks and tended to reduce body weight at 9-12 weeks (not significant) compared with the HFD control group. Supplementation with 0.4% HT077 and 0.02% orlistat significantly decreased body weights at 4-12 and 1-12 weeks, respectively, compared with those in the HFD control group. At the end of the experiment, mice fed an HFD containing 0.4% HT077 showed markedly reduced body weight gains compared with those in the HFD control mice (both p < 0.001, Figure 1b). HFD-fed control mice showed significantly lower food intake (weeks 0-4 and 8-12) or lower food intake (weeks 4-8) than ND-fed mice during the study period ( Figure 1c). HFD-fed control mice had significantly higher energy intake (weeks 4-12) or higher energy intake (weeks 0-4) than ND-fed mice (Figure 1d). There was no significant difference in food intake and energy intake between the HFD + HT077 and HFD control groups. The food and energy intakes of the HFD + 0.02% orlistat group were significantly higher than those of the HFD control group for 12 weeks. In addition, no abnormal clinical signs of toxicity were observed in the HFD + HT077 group during the experimental period.

Effects of HT077 on Abdominal Fat, Liver, and Spleen Wet Weights
The absolute and relative weights of total abdominal, perirenal, and mesenteric adipose tissue and the absolute epididymal adipose tissue weight were significantly increased in the HFD control group compared with those in the ND group, showing that 12 weeks of HFD feeding caused obesity Effects of an extract mixture of Prunus persica and Nelumbo nucifera (HT077) on body weight, and food and energy consumption in C57BL/6 mice fed a high-fat diet (HFD). (a) Body weight changes in mice that received either a normal diet (ND), HFD, HFD containing 0.02% orlistat, or HFD containing 0.1, 0.2, or 0.4% HT077 for 12 weeks. (b) Body weight gain for 12 weeks. Changes in (c) food intake and (d) energy intake over 12 weeks. All values are presented as the mean ± SD (n = 8 for ND, 16 for HFD, and 12 for the other groups). # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. ND group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. HFD control group.

Effects of HT077 on Abdominal Fat, Liver, and Spleen Wet Weights
The absolute and relative weights of total abdominal, perirenal, and mesenteric adipose tissue and the absolute epididymal adipose tissue weight were significantly increased in the HFD control group compared with those in the ND group, showing that 12 weeks of HFD feeding caused obesity (all p < 0.001, Figure 2a-h). In contrast, supplementation with HT077 and orlistat significantly reduced the absolute and relative weights of perirenal, mesenteric, and total abdominal fat pad compared with those in the HFD control group (Figure 2c-h). HFD feeding increased the absolute liver weights compared with the those in the ND group (p < 0.001, Figure 2i), whereas supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 significantly reduced the absolute and relative liver weights compared with those in the HFD group (all p < 0.001, Figure 2i,j). No difference was observed in spleen weights among the groups (Figure 2k,l).
Nutrients 2020, 12, x FOR PEER REVIEW 6 of 18 (all p < 0.001, Figure 2a-h). In contrast, supplementation with HT077 and orlistat significantly reduced the absolute and relative weights of perirenal, mesenteric, and total abdominal fat pad compared with those in the HFD control group (Figure 2c-h). HFD feeding increased the absolute liver weights compared with the those in the ND group (p < 0.001, Figure 2i), whereas supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 significantly reduced the absolute and relative liver weights compared with those in the HFD group (all p < 0.001, Figure 2i,j). No difference was observed in spleen weights among the groups (Figure 2k,l).   Figure 2. Effects of an extract mixture of Prunus persica and Nelumbo nucifera (HT077) on the absolute and relative weights of abdominal fat (a-h), liver (i-j), and spleen (k-l) in C57BL/6 mice fed a highfat diet (HFD). The absolute wet weights of white adipose tissues isolated from visceral regions including epididymal (a), perirenal (c), and mesenteric (e) deposits were measured. Total abdominal fat weights (g) are the sums of three individual fat depots (a,c,e). The relative organ weight was calculated with respect to fasted body weight. Mice received either a normal diet (ND), HFD, HFD containing 0.02% orlistat, or HFD containing 0.1, 0.2, or 0.4% HT077 for 12 weeks. All values are presented as the mean ± SD (n = 8 for ND, 16 for HFD, and 12 for the other groups). ### p < 0.001 vs. ND group; * p < 0.05 and ** p < 0.01, *** p < 0.001 vs. HFD control group. BW, body weight.

Figure 2.
Effects of an extract mixture of Prunus persica and Nelumbo nucifera (HT077) on the absolute and relative weights of abdominal fat (a-h), liver (i-j), and spleen (k-l) in C57BL/6 mice fed a high-fat diet (HFD). The absolute wet weights of white adipose tissues isolated from visceral regions including epididymal (a), perirenal (c), and mesenteric (e) deposits were measured. Total abdominal fat weights (g) are the sums of three individual fat depots (a,c,e). The relative organ weight was calculated with respect to fasted body weight. Mice received either a normal diet (ND), HFD, HFD containing 0.02% orlistat, or HFD containing 0.1, 0.2, or 0.4% HT077 for 12 weeks. All values are presented as the mean ± SD (n = 8 for ND, 16 for HFD, and 12 for the other groups). ### p < 0.001 vs. ND group; * p < 0.05 and ** p < 0.01, *** p < 0.001 vs. HFD control group. BW, body weight.

Effects of HT077 on Serum Levels of Glucose, Lipids, and Liver Enzymes
HFD feeding significantly increased serum glucose levels compared with those in the ND group (p < 0.001, Figure 3a). In contrast, supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 significantly reduced glucose levels compared with those in the HFD control group Nutrients 2020, 12, 3392 8 of 18 (all p < 0.001). No significant differences were observed in triglyceride levels among the groups ( Figure 3b). Total cholesterol levels were not altered by HFD feeding but were significantly reduced by supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 compared with those in the HFD control group (Figure 3c). Likewise, serum AST levels were not altered by HFD feeding but were significantly reduced by supplementation with 0.02% orlistat and 0.4% HT077 compared with those in the HFD control group (both p < 0.001, Figure 3d). The HFD significantly elevated serum ALT levels and decreased the AST/ALT ratio compared with those in the ND group (both p < 0.001, Figure 3e,f), whereas supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 reversed these changes.

Effects of HT077 on Serum Levels of Glucose, Lipids, and Liver Enzymes
HFD feeding significantly increased serum glucose levels compared with those in the ND group (p < 0.001, Figure 3a). In contrast, supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 significantly reduced glucose levels compared with those in the HFD control group (all p < 0.001). No significant differences were observed in triglyceride levels among the groups (Figure 3b). Total cholesterol levels were not altered by HFD feeding but were significantly reduced by supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 compared with those in the HFD control group (Figure 3c). Likewise, serum AST levels were not altered by HFD feeding but were significantly reduced by supplementation with 0.02% orlistat and 0.4% HT077 compared with those in the HFD control group (both p < 0.001, Figure 3d). The HFD significantly elevated serum ALT levels and decreased the AST/ALT ratio compared with those in the ND group (both p < 0.001, Figure 3e,f), whereas supplementation with 0.02% orlistat and 0.1%, 0.2%, and 0.4% HT077 reversed these changes.

Effects of HT077on Serum Adipokine Concentrations
Adiponectin levels tended to decrease after HFD feeding (statistically not significant), but significantly increased with 0.02% orlistat and 0.2% and 0.4% HT077 supplementation, as compared with levels in the HFD control group (Figure 4a). HFD feeding significantly increased serum leptin levels and decreased the adiponectin/leptin ratio compared with those in the ND group (p < 0.001, Figure 4b, c), whereas supplementation with 0.02% orlistat and 0.4% HT077 significantly reversed these changes.

Effects of HT077on Serum Adipokine Concentrations
Adiponectin levels tended to decrease after HFD feeding (statistically not significant), but significantly increased with 0.02% orlistat and 0.2% and 0.4% HT077 supplementation, as compared with levels in the HFD control group (Figure 4a). HFD feeding significantly increased serum leptin levels and decreased the adiponectin/leptin ratio compared with those in the ND group (p < 0.001, Figure 4b, c), whereas supplementation with 0.02% orlistat and 0.4% HT077 significantly reversed these changes.

Effects of HT077on Hepatic Triglyceride and Total Cholesterol Contents
Twelve weeks of HFD feeding significantly increased triglyceride levels in the liver (p < 0.05, Figure 5a) but did not change total cholesterol levels ( Figure 5b). Hepatic triglyceride levels were significantly reduced by supplementation with 0.4% HT077 (p < 0.05) but not 0.02% orlistat. Hepatic total cholesterol levels were significantly decreased by supplementation with 0.4% HT077 and 0.02% orlistat (both p < 0.01).

Effects of HT077on Hepatic Triglyceride and Total Cholesterol Contents
Twelve weeks of HFD feeding significantly increased triglyceride levels in the liver (p < 0.05, Figure 5a) but did not change total cholesterol levels ( Figure 5b). Hepatic triglyceride levels were significantly reduced by supplementation with 0.4% HT077 (p < 0.05) but not 0.02% orlistat. Hepatic total cholesterol levels were significantly decreased by supplementation with 0.4% HT077 and 0.02% orlistat (both p < 0.01).

Effects of HT077on Lipogenesis and Fatty Acid Oxidation-Related Gene Expression in the Adipose Tissue
To explore the possible mechanisms underlying the observed effects of HT077, gene expression related to lipid metabolism in mesenteric adipose tissue was compared between HFD and HFD + HT077 groups. Since a high dose (0.4%) was the most effective at reducing adiposity, the HFD + 0.4% HT077 group was chosen for analysis. HT077 tended to decrease the expression levels of SREBP-1c in mesenteric adipose tissue compared with those in the HFD control mice (p = 0.0574, Figure 6a). The mRNA levels of FAS and SCD-1 were significantly decreased in the HFD + 0.4% HT077 group compared with those in the HFD control group (p < 0.001 and p < 0.01, respectively), whereas there was no difference in ACC mRNA levels between the two groups. The mRNA levels of PGC-1a and PPARα were significantly increased in the HFD + 0.4% HT077 group compared with those in the HFD control group (both p < 0.05, Figure 6b), whereas CPT1 mRNA levels were not different between the two groups. mRNA levels of FAS and SCD-1 were significantly decreased in the HFD + 0.4% HT077 group compared with those in the HFD control group (p < 0.001 and p < 0.01, respectively), whereas there was no difference in ACC mRNA levels between the two groups. The mRNA levels of PGC-1a and PPARα were significantly increased in the HFD + 0.4% HT077 group compared with those in the HFD control group (both p < 0.05, Figure 6b), whereas CPT1 mRNA levels were not different between the two groups.

Discussion
This study revealed that the herbal mixture of P. persica and N. nucifera, which are commonly used as teas for weight loss in East Asia, reduced body weight and fat accumulation in the adipose tissue and livers of obese mice. In addition, HT077 improved circulating glucose, lipid, and adipokine profiles and regulated the expression of genes involved in lipid metabolism in adipose tissue.
HT077 reduced body weight and visceral fat in HFD-fed mice without noticeable changes in food and energy consumption. HFD-fed rodents had increased caloric consumption and decreased food intake compared with ND-fed rodents, which is possibly due to impaired satiation signaling, and leads to fat accumulation and weight gain [25]. It is well established that visceral fat is the main contributor to abdominal obesity and is related to an increased risk of metabolic dysfunction. Representative visceral fat deposits in rodents include those of the epididymal, perirenal, and mesenteric fat pad [26]. Among these, the accumulation of perirenal fat increases sodium reabsorption due to kidney compression and activates the renin-angiotensin-aldosterone system, causing the development of hypertension, insulin resistance, and atherosclerosis [27][28][29]. Mesenteric fat drained through portal circulation has higher lipolysis activities than nonportal adipose tissue, and its excessive accumulation is expected to increase free fatty acid flux to the liver, leading to fatty liver and insulin resistance [30,31]. Notably, HT077 at a high dose (0.4%) reduced the perirenal and mesenteric fat weights by 61.4 and 74.4%, respectively, compared with those in the control group. HT077 did not affect food and energy consumption, whereas orlistat, a lipase inhibitor, increased both parameters. In line with our results, intestinal lipase inhibition has been shown to increase appetite and food consumption, which may counteract the therapeutic effects of orlistat [32]. Our results indicate that HT077 exerts promising anti-obesity effects in the absence of a change in appetite.
Long-term HFD intake causes ectopic fat deposition in several organs other than adipose tissue, such as liver and spleen. HFD feeding for 12 weeks induced a significant increase in liver weight but not in spleen weight, which is consistent with previous results [33]. HT077 inhibited hepatic fat accumulation and liver weight gain in HFD-fed mice, along with decreased ALT and AST levels and an increased AST/ALT ratio. Hepatic fat accumulation arises from increased circulating fatty acid uptake and decreased β-oxidation in the liver, leading to NAFLD [34]. Furthermore, increased hepatic fat is known as an independent indicator of obesity-related metabolic complications [35]. NAFLD following HFD is denoted by the elevated leakage of liver enzymes from hepatocytes into the blood, with that of ALT being more prominent than that of AST, and by an AST/ALT ratio less than 1 [36,37], all of which are consistent with our results. P. persica and N. nucifera are known to inhibit fatty liver induced by HFD in animals by regulating hepatic lipogenesis [8,[38][39][40]. Together with these previous findings, our results suggest that HT077 might prevent obesity-related fatty liver diseases.
Obesity is commonly a co-morbidity of glucose and lipid metabolism disorders. HFD consumption increases hepatic glucose output and decreases glucose uptake in peripheral tissues, resulting in hyperglycemia [41]. This increases insulin secretion and consequently activates hepatic lipogenesis and cholesterol excretion, leading to hyperlipidemia [41]. In this study, the serum levels of glucose and total cholesterol were significantly reduced after HT077 supplementation, indicating hypoglycemic and hypocholesterolemic activities. Previous studies have shown that P. persica and its major component multiflorin A reduce blood glucose levels in HFD-fed and glucose-loaded mice, respectively [8,42]. Further, N. nucifera improves glucose tolerance and insulin resistance in rodents fed an HFD [43,44] and shows anti-hyperglycemic activity in diabetic rats [45]. Moreover, we previously reported the synergistic effects of combined P. persica and N. nucifera on hypoglycemic activity in HFD-induced obese mice [9]. In addition, N. nucifera and its total flavonoids are known to improve lipid profiles of rats fed an HFD [18,46]. Our results, along with those of previous studies, suggest that HT077 might improve obesity-associated hyperglycemia and hypercholesterolemia.
Over the past few decades, it has been established that adipose tissue is not simply an energy reservoir but is an endocrine organ that secretes adipokines such as leptin and adiponectin. Leptin induces weight and fat loss by suppressing appetite and elevating energy expenditure through action in the hypothalamus [47]. Leptin resistance, marked by hyperleptinemia, occurs universally in human obesity and is a key feature of HFD-induced obesity in rodents [48]. It is known to exacerbate adiposity and hepatic triglyceride accumulation and impair the homeostasis of peripheral glucose and lipid metabolism [49]. Adiponectin has been shown to regulate peripheral glucose and fatty acid metabolism by increasing the insulin sensitivity of target organs, including the liver and muscle [50]. In obesity and metabolic syndrome, circulating adiponectin levels decrease and contribute to the development of insulin resistance [50]. In addition, the adiponectin/leptin ratio has recently been proposed as an indicator of adipose tissue dysfunction [51]. An increase in this ratio is better correlated with reduced insulin resistance than leptin or adiponectin alone [52]. Herein, HT077 significantly decreased circulating leptin levels and increased adiponectin levels and the adiponectin/leptin ratio. In support of our results, nuciferine, a major active alkaloid in N. nucifera leaf, reportedly reduces leptin and increases adiponectin levels in HFD-fed hamsters [53]. Our results indicate that improvements in leptin and adiponectin levels following HT077 administration might contribute to counteracting HFD-induced excessive lipid accumulation and abnormal glucose and lipid metabolism.
Adipose tissue plays an important role in the regulation of lipid metabolism and its metabolic functions include lipogenesis and fatty acid oxidation [54]. To better understand the mechanisms underlying the effects of HT077, we analyzed gene expression related to lipid metabolism in mesenteric adipose tissue. Mesenteric fat is known to be the most similar visceral tissue between humans and rodents because of its high vascularity and portal venous drainage [55,56], and in this study, was the site where lipid accumulation was most inhibited by HT077. In C57BL/6 mice, HFD affects lipid metabolism in adipose tissue differently depending on the site of adipose tissue [57,58]. In mesenteric adipose tissue, lipogenic genes such as SREBP-1c, ACC, FAS, and SCD-1 are upregulated by an HFD compared with a ND, and inhibition of this upregulation exerts anti-obesity effects [58][59][60][61]. The expression of FAS and SCD-1 was significantly decreased by HT077 administration compared with that in the control mice. FAS and SCD-1 are central enzymes in de novo lipogenesis in adipose tissue [62]. FAS catalyzes the biosynthesis of saturated long-chain fatty acids from acetyl and malonyl coenzyme A and NADPH, whereas SCD-1 catalyzes the biosynthesis of monounsaturated fatty acids from their saturated fatty acid precursors [62]. Given that increased expression and activity of FAS and SCD-1 in adipose tissue contribute to the development of obesity and insulin resistance [63,64], decreased lipid synthesis through the inhibition of FAS and SCD-1 expression might contribute to the inhibitory effects of HT077 on adiposity and related metabolic disorders. Furthermore, an HFD upregulates the expression of fatty acid oxidation-related genes such as PGC-1α, PPARα, and CPT-1 in C57BL/6 mice and this upregulation attenuates fat accumulation in adipose tissue and liver [57,58]. HT077 increased the expression of PGC-1α and PPARα compared with that in the control mice. PGC-1α and PPAR-α are key regulators of mitochondrial biogenesis. Their overexpression in adipocytes induces the expression of target genes involved in fatty acid oxidation, thereby inhibiting fat accumulation and improving insulin resistance [65,66]. Increased fatty acid oxidation in mesenteric fat induced by HT077 might contribute to the inhibition of free fatty acid influx into the liver, suppressing the development of fatty liver. Overall, our results suggest that the anti-obesity effects of HT077 are at least partially related to decreased lipogenesis and increased fatty acid oxidation in adipose tissue.
HT077 showed dose-dependent effects in reducing abdominal and hepatic fat accumulation and improving adipokine imbalances. Notably, HT077 at a high dose (0.4%) inhibited fat accumulation similarly to orlistat. Glucose and total cholesterol levels were lowered similarly by all doses of HT077, and in particular, the hypoglycemic efficacy was comparable to that of orlistat. Our results overall suggest that high-dose HT077 therapy may provide beneficial effects as orlistat does. When calculated based on the food intake and body weight, the average daily doses of 0.1, 0.2, and 0.4% HT077 groups are approximately 90, 180, and 360 mg/kg of mouse body weight, respectively, and that of 0.02% orlistat group is 25 mg/kg. The mouse dose of orlistat corresponds to a human equivalent dose of about 120 mg/day for 60 kg person based on body surface area [67]. Considering that 90-180 mg/day dosage of orlistat resulted in an 8.5-8.8% reduction of initial body weight in a 6-month dose-ranging study of obese subjects [68], HT077 has a favorable anti-obesity efficacy.
Given that HT077 decreased FAS and SCD-1 expression and increased PGC-1α and PPARα expression in white adipose tissue (WAT), it is tempting to speculate that HT077 prevents HFD-induced weight gain by increasing energy expenditure via induction of WAT browning. Previous studies have shown that targeted deletion of FAS in adipose tissue induces brown fat-like adipocytes in WAT, increases energy expenditure, and decreases diet-induced obesity [69]. SCD-1 deficiency potentiates beige adipocyte formation and increases energy expenditure in HFD-fed mice [70]. In addition, overexpression of PGC-1α and PPARα induces brown fat feature in white adipocytes [71]. As browning of white fat significantly contributes to an increase in whole-body energy expenditure [72], HT077 is hypothesized to increase energy expenditure and this hypothesis requires further investigation to measure the energy expenditure of animals using metabolic cages, which was not examined in this study. In addition, the effects of HT077 on obesity-induced insulin resistance could be investigated since Nelumbo nucifera and its active constituent nuciferine have been shown to decrease serum insulin levels and homeostasis model assessment for insulin resistance index in HFD-induced obese rodents [44,53].

Conclusions
Our results suggest that HT077 has anti-obesity effects and inhibits the development of obesity-related metabolic disorders such as fatty liver, hyperglycemia, and hyperlipidemia. These effects might be partially mediated by an improvement in blood leptin and adiponectin concentrations and the regulation of lipid synthesis and fatty acid oxidation in adipose tissue. Our results support the potential health benefits of HT077 in managing obesity and related metabolic diseases.